Method, array and use for determining the presence of pancreatic cancer

ABSTRACT

The present invention relates to a method for determining the presence of pancreatic cancer in an individual comprising or consisting of the steps of: (a) providing a sample to be tested from the individual, and (b) determining a biomarker signature of the test sample by measuring the expression in the test sample of one or more biomarkers selected from the group defined in Table III, wherein the expression in the test sample of one or more biomarkers selected from the group defined in Table III is indicative of the individual having pancreatic cancer. The invention also comprises arrays and kits of parts for use in the method of the invention.

FIELD OF INVENTION

The present invention relates to methods for diagnosis of pancreatic cancer, and biomarkers and arrays for use in the same.

BACKGROUND

Despite major efforts, pancreatic cancer (PaC) still carries a poor prognosis [1]. While PaC is only the 10^(th) most common cancer, it is the 4^(th) leading cause of cancer death in the USA [2-4]. In fact, the 5-year survival is <5%, the lowest of all malignancies [2-3]. However, recent data have shown that the outcome could be dramatically improved by early detection when the cancer is still predominantly at stage I, as illustrated by a 5-year survival of 30-60% (≤20 mm sized tumour) and even >75% (≤10 mm sized tumour) after early PaC resection [2-4].

PaC is characterized by a rapid tumour progression, early metastasization, and unresponsiveness to most conventional therapies [1, 5]. The poor prognosis is mainly due to the lack of effective early diagnostics combined with that disease-specific clinical symptoms occur late in the course of the disease. At the time of diagnosis, the tumour has often reached a size of 30-40 mm and a majority of all patients (52%) already have metastases, 26% locally advanced cancer, and only 7% have tumours confined to the pancreas [2, 4]. At this time, about 15% of the patients are still operable, but their median survival is only 20 months.

A variety of non-invasive methodologies, including (endoscopic) ultrasound, computed tomography, and/or endoscopic retrograde cholangio-pancreatography, are used for PaC diagnostics [1-2, 6]. Albeit powerful, these methods are not specific for PaC and not designed for early detection when the tumour is still small and potentially curable. The situation is further complicated by the fact that PaC is difficult to differentiate from benign conditions, such as chronic pancreatitis, using currently available diagnostic tools [2]. Hence, the use of biomarkers for specific and early detection of PaC would be of invaluable clinical benefit.

In spite of major efforts, molecular fingerprints associated with PaC from in particular, crude, non-fractionated serum and plasma, remains to be deciphered [2, 7-9]. Among the number of mainly single biomarkers that have been outlined so far, including e.g. CRP, CA 242, GDF-15, haptoglobin, M2-pyruvate kinase, serum amyloid A, IGFBP-1, none have proven to be clinically superior to CA 19-9 [2, 8-10]. Still, the use of CA 19-9 is significantly hampered by the fact that it has been found to i) be elevated in both non-malignant conditions (e.g. pancreatitis and acute cholangitis) and other gastro-intestinal cancers (e.g. gastric cancer and colorectal cancer), ii) lack sensitivity for early PaC, and iii) be absent in about 10% of the population [2, 8-10]. When screening for PaC, CA 19-9 has only yielded medium sensitivity (ranging from 69% to 98%) and specificity (46% to 98%) [2, 9-11].

Against this background, the inventors developed a proteomic approach to prognostic diagnosis of cancer in WO 2008/117067 whereby the first sets of serum biomarkers for detection of pancreatic cancer and for predicting survival were identified.

SUMMARY OF THE INVENTION

Motivated by a recent study, in which we indicated that affinity proteomics [12-13] could be used to pin-point candidate PaC serum biomarker signatures [14], we have further deciphered the serum proteome of PaC.

In this study, we have for the first time pre-validated multiplexed serum biomarker signatures for PaC diagnosis, demonstrating that diagnostic information could be extracted from crude blood samples, displaying high specificity and sensitivity. This provides enhanced PaC diagnosis and thereby improved prognosis, bringing significantly added clinical value, as well as shedding further light on the underlying, intricate disease biology.

Accordingly, a first aspect of the invention provides a method for determining the presence of pancreatic cancer in an individual comprising or consisting of the steps of:

-   -   a) providing a sample to be tested from the individual;     -   b) determining a biomarker signature of the test sample by         measuring the expression in the test sample of one or more         biomarkers selected from the group defined in Table III;         wherein the expression in the test sample of one or more         biomarkers selected from the group defined in Table III is         indicative of the individual having pancreatic cancer.

By “sample to be tested”, “test sample” or “control sample” we include tissue, fluid proteome and/or expressome samples from an individual to be tested or a control individual, as appropriate.

By “expression” we mean the level or amount of a gene product such as mRNA or protein.

Methods of detecting and/or measuring the concentration of protein and/or nucleic acid are well known to those skilled in the art, see for example Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press.

By “biomarker” we mean a naturally-occurring biological molecule, or component or fragment thereof, the measurement of which can provide information useful in the prognosis of pancreatic cancer. For example, the biomarker may be a naturally-occurring protein or carbohydrate moiety, or an antigenic component or fragment thereof.

In one embodiment, the method comprises or consists of steps (a) and (b) and the further steps of:

-   -   c) providing a control sample from an individual not afflicted         with pancreatic cancer (i.e. a negative control);     -   d) determining a biomarker signature of the control sample by         measuring the expression in the control sample of the one or         more biomarkers measured in step (b);         wherein the presence of pancreatic cancer is identified in the         event that the expression in the test sample of the one or more         biomarkers measured in step (b) is different from the expression         in the control sample of the one or more biomarkers measured in         step (d).

In another embodiment, the method comprises or consists of steps (a), (b), (c) and (d) and the additional steps of:

-   -   e) providing a control sample from an individual afflicted with         pancreatic cancer (i.e. a positive control);     -   f) determining a biomarker signature of the control sample by         measuring the expression in the control sample of the one or         more biomarkers measured in step (b);         wherein the presence of pancreatic cancer is identified in the         event that the expression in the test sample of the one or more         biomarkers measured in step (b) corresponds to the expression in         the control sample of the one or more biomarkers measured in         step (f).

By “corresponds to the expression in the control sample” we include that the expression of the one or more biomarkers in the sample to be tested is the same as or similar to the expression of the one or more biomarkers of the positive control sample. Preferably the expression of the one or more biomarkers in the sample to be tested is identical to the expression of the one or more biomarkers of the positive control sample.

Differential expression (up-regulation or down regulation) of biomarkers, or lack thereof, can be determined by any suitable means known to a skilled person. Differential expression is determined to a p value of a least less than 0.05 (p=<0.05), for example, at least <0.04, <0.03, <0.02, <0.01, <0.009, <0.005, <0.001, <0.0001, <0.00001 or at least <0.000001. Preferably, differential expression is determined using a support vector machine (SVM). Preferably, the SVM is an SVM as described below. Most preferably, the SVM is the SVM described in Table V(A), below.

It will be appreciated by persons skilled in the art that differential expression may relate to a single biomarker or to multiple biomarkers considered in combination (i.e. as a biomarker signature). Thus, a p value may be associated with a single biomarker or with a group of biomarkers. Indeed, proteins having a differential expression p value of greater than 0.05 when considered individually may nevertheless still be useful as biomarkers in accordance with the invention when their expression levels are considered in combination with one or more other biomarkers.

In one embodiment, step (b) comprises or consists of measuring the expression of one or more of the biomarkers listed in Table IV(A), for example, at least 2 of the biomarkers listed in Table IV(A).

As exemplified in the accompanying examples, the expression of certain proteins in a blood, serum or plasma test sample may be indicative of pancreatic cancer in an individual. For example, the relative expression of certain serum proteins in a single test sample may be indicative of the presence of pancreatic cancer in an individual.

Preferably, the individual is a human. However, the individual being tested may be any mammal, such as a domesticated mammal (preferably of agricultural or commercial significance including a horse, pig, cow, sheep, dog and cat).

Preferably, step (b) comprises or consists of measuring the expression of interleukin-7 (IL-7) and/or integrin alpha-10, for example, measuring the expression of interleukin-7, measuring the expression of integrin alpha-10, or measuring the expression of interleukin-7 and integrin alpha-10. Most preferably, step (b) comprises or consists of measuring the expression of each the biomarkers listed in Table IV(A).

In one embodiment, step (b) comprises or consists of measuring the expression of 1 or more biomarkers from the biomarkers listed in Table IV(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the biomarkers listed in Table IV(B). Hence, step (b) preferably comprises or consists of measuring the expression of all of the biomarkers listed in Table IV(B).

In another embodiment, step (b) comprises or consists of measuring the expression of 1 or more biomarkers from the biomarkers listed in Table IV(C), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 of the biomarkers listed in Table IV(C). Preferably, step (b) comprises or consists of measuring the expression of all of the biomarkers listed in Table IV(C).

Preferably, step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers defined in Table IV.

In one embodiment, the method is for differentiating between pancreatic cancer (PaC) and another disease state.

Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A). Preferably, step (b) also comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B). It is also preferred that step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in Table V(C).

Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(D), for example at least 2 or 3 of the biomarkers listed in Table V(D). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(F), for example at least 2, 3, 4, 5 or 6 of the biomarkers listed in Table V(F). Preferably, step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(C), Table V(D) and/or Table V(F).

By “differentiating between pancreatic cancer (PaC) and another disease state” we include differentiating between PaC and any other condition, including a state of health.

In one embodiment, the other disease state or states is chronic pancreatitis (ChP), acute inflammatory pancreatitis (AIP) and/or normal, for example, the other disease state or states may be chronic pancreatitis alone; acute inflammatory pancreatitis alone; chronic pancreatitis and acute inflammatory pancreatitis; chronic pancreatitis and normal; acute inflammatory pancreatitis and normal; or, chronic pancreatitis, acute inflammatory pancreatitis and normal.

When referring to a “normal” disease state we include individuals not afflicted with chronic pancreatitis (ChP) or acute inflammatory pancreatitis (AIP). Preferably the individuals are not afflicted with any pancreatic disease or disorder. Most preferably, the individuals are healthy individuals, i.e., they are not afflicted with any disease or disorder.

In a another embodiment, the method is for differentiating between pancreatic cancer and chronic pancreatitis (ChP). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A). Step (b) may comprise or consist of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in Table V(C). Step (b) may comprise or consist of measuring the expression in the test sample of all of the biomarkers listed in Table V(A) and/or Table V(C).

In an additional/alternative embodiment, the method is for differentiating between pancreatic cancer and acute inflammatory pancreatitis (AIP) and step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B). Step (b) may comprise or consist of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in Table V(C). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(E). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(F), for example at least 2, 3, 4, 5 or 6 of the biomarkers listed in Table V(F). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(H), for example at least 2 or 3 of the biomarkers listed in Table V(H). Hence, step (b) preferably comprises or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(C), Table V(E), Table V(F) and/or Table IV(H).

In one embodiment, the method is for differentiating between pancreatic cancer and normal (N). For a definition of “normal” disease state, see above. Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B). Preferably, step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(D), for example at least 2 or 3 of the biomarkers listed in Table V(D). Preferably, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(E). It is also preferred that step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(G), for example at least 2 or 3 of the biomarkers listed in Table V(G). Hence, step (b) may comprise or consist of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(D), Table V(E) and/or Table IV(G).

In one embodiment, step (b) comprises or consists of measuring the expression of IL-3. In a further embodiment, step (b) comprises or consists of measuring the expression of Integrin α-10. In a still further embodiment, step (b) comprises or consists of measuring the expression of Mucin-1. In another embodiment, step (b) comprises or consists of measuring the expression of C1s. In an additional embodiment, step (b) comprises or consists of measuring the expression of MCP-3. In one embodiment, step (b) comprises or consists of measuring the expression of Angiomotin. In a further embodiment, step (b) comprises or consists of measuring the expression of BTK. In a still further embodiment, step (b) comprises or consists of measuring the expression of C1q. In another embodiment, step (b) comprises or consists of measuring the expression of CD40 ligand.

In an additional embodiment, step (b) comprises or consists of measuring the expression of GM-CSF. In one embodiment, step (b) comprises or consists of measuring the expression of IgM. In a further embodiment, step (b) comprises or consists of measuring the expression of IL-11. In a still further embodiment, step (b) comprises or consists of measuring the expression of IL-16. In another embodiment, step (b) comprises or consists of measuring the expression of IL-1-ra. In an additional embodiment, step (b) comprises or consists of measuring the expression of IL-1α. In one embodiment, step (b) comprises or consists of measuring the expression of IL-1β. In a further embodiment, step (b) comprises or consists of measuring the expression of IL-2. In a still further embodiment, step (b) comprises or consists of measuring the expression of IL-7. In another embodiment, step (b) comprises or consists of measuring the expression of IL-9. In an additional embodiment, step (b) comprises or consists of measuring the expression of INF-γ. In one embodiment, step (b) comprises or consists of measuring the expression of Integrin α-11. In a further embodiment, step (b) comprises or consists of measuring the expression of JAK3. In a still further embodiment, step (b) comprises or consists of measuring the expression of Leptin. In another embodiment, step (b) comprises or consists of measuring the expression of Lewis y. In an additional embodiment, step (b) comprises or consists of measuring the expression of MCP-4. In one embodiment, step (b) comprises or consists of measuring the expression of Procathepsin W. In a further embodiment, step (b) comprises or consists of measuring the expression of Properdin. In a still further embodiment, step (b) comprises or consists of measuring the expression of PSA. In another embodiment, step (b) comprises or consists of measuring the expression of RANTES. In an additional embodiment, step (b) comprises or consists of measuring the expression of Sialyl Lewis x. In one embodiment, step (b) comprises or consists of measuring the expression of TM peptide. In a further embodiment, step (b) comprises or consists of measuring the expression of TNF-α. In a still further embodiment, step (b) comprises or consists of measuring the expression of C4. In another embodiment, step (b) comprises or consists of measuring the expression of β-galactosidase.

In an additional embodiment, step (b) comprises or consists of measuring the expression of IL-12. In one embodiment, step (b) comprises or consists of measuring the expression of TGF-β1. In a further embodiment, step (b) comprises or consists of measuring the expression of VEGF. In a still further embodiment, step (b) comprises or consists of measuring the expression of IL-8. In another embodiment, step (b) comprises or consists of measuring the expression of C3. In an additional embodiment, step (b) comprises or consists of measuring the expression of IFN-γ. In one embodiment, step (b) comprises or consists of measuring the expression of IL-10. In a further embodiment, step (b) comprises or consists of measuring the expression of IL-13. In a still further embodiment, step (b) comprises or consists of measuring the expression of IL-18. In another embodiment, step (b) comprises or consists of measuring the expression of IL-6. In an additional embodiment, step (b) comprises or consists of measuring the expression of Lewis x. In one embodiment, step (b) comprises or consists of measuring the expression of Eotaxin. In a further embodiment, step (b) comprises or consists of measuring the expression of C1 esterase inhibitor. In a still further embodiment, step (b) comprises or consists of measuring the expression of MCP-1. In another embodiment, step (b) comprises or consists of measuring the expression of TNF-β. In an additional embodiment, step (b) comprises or consists of measuring the expression of GLP-1. In one embodiment, step (b) comprises or consists of measuring the expression of IL-5. In a further embodiment, step (b) comprises or consists of measuring the expression of IL-4. In a still further embodiment, step (b) comprises or consists of measuring the expression of Factor B. In another embodiment, step (b) comprises or consists of measuring the expression of C5. In an additional embodiment, step (b) comprises or consists of measuring the expression of CD40.

In one embodiment, step (b) does not comprise measuring the expression of IL-3. In a further embodiment, step (b) does not comprise measuring the expression of Integrin α-10. In a still further embodiment, step (b) does not comprise measuring the expression of Mucin-1. In another embodiment, step (b) does not comprise measuring the expression of C1s. In an additional embodiment, step (b) does not comprise measuring the expression of MCP-3. In one embodiment, step (b) does not comprise measuring the expression of Angiomotin. In a further embodiment, step (b) does not comprise measuring the expression of BTK. In a still further embodiment, step (b) does not comprise measuring the expression of C1q. In another embodiment, step (b) does not comprise measuring the expression of CD40 ligand. In an additional embodiment, step (b) does not comprise measuring the expression of GM-CSF. In one embodiment, step (b) does not comprise measuring the expression of IgM. In a further embodiment, step (b) does not comprise measuring the expression of IL-11. In a still further embodiment, step (b) does not comprise measuring the expression of IL-16. In another embodiment, step (b) does not comprise measuring the expression of IL-1-ra. In an additional embodiment, step (b) does not comprise measuring the expression of IL-1α. In one embodiment, step (b) does not comprise measuring the expression of IL-1β. In a further embodiment, step (b) does not comprise measuring the expression of IL-2. In a still further embodiment, step (b) does not comprise measuring the expression of IL-7. In another embodiment, step (b) does not comprise measuring the expression of IL-9. In an additional embodiment, step (b) does not comprise measuring the expression of INF-γ. In one embodiment, step (b) does not comprise measuring the expression of Integrin α-11. In a further embodiment, step (b) does not comprise measuring the expression of JAK3. In a still further embodiment, step (b) does not comprise measuring the expression of Leptin. In another embodiment, step (b) does not comprise measuring the expression of Lewis y. In an additional embodiment, step (b) does not comprise measuring the expression of MCP-4. In one embodiment, step (b) does not comprise measuring the expression of Procathepsin W. In a further embodiment, step (b) does not comprise measuring the expression of Properdin. In a still further embodiment, step (b) does not comprise measuring the expression of PSA. In another embodiment, step (b) does not comprise measuring the expression of RANTES. In an additional embodiment, step (b) does not comprise measuring the expression of Sialyl Lewis x. In one embodiment, step (b) does not comprise measuring the expression of TM peptide. In a further embodiment, step (b) does not comprise measuring the expression of TNF-α. In a still further embodiment, step (b) does not comprise measuring the expression of C4.

In another embodiment, step (b) does not comprise measuring the expression of β-galactosidase.

In an additional embodiment, step (b) does not comprise measuring the expression of IL-12. In one embodiment, step (b) does not comprise measuring the expression of TGF-β1. In a further embodiment, step (b) does not comprise measuring the expression of VEGF. In a still further embodiment, step (b) does not comprise measuring the expression of IL-8. In another embodiment, step (b) does not comprise measuring the expression of C3. In an additional embodiment, step (b) does not comprise measuring the expression of IFN-γ. In one embodiment, step (b) does not comprise measuring the expression of IL-10. In a further embodiment, step (b) does not comprise measuring the expression of IL-13. In a still further embodiment, step (b) does not comprise measuring the expression of IL-18. In another embodiment, step (b) does not comprise measuring the expression of IL-6. In an additional embodiment, step (b) does not comprise measuring the expression of Lewis x. In one embodiment, step (b) does not comprise measuring the expression of Eotaxin. In a further embodiment, step (b) does not comprise measuring the expression of C1 esterase inhibitor. In a still further embodiment, step (b) does not comprise measuring the expression of MCP-1. In another embodiment, step (b) does not comprise measuring the expression of TNF-β. In an additional embodiment, step (b) does not comprise measuring the expression of GLP-1. In one embodiment, step (b) does not comprise measuring the expression of IL-5. In a further embodiment, step (b) does not comprise measuring the expression of IL-4. In a still further embodiment, step (b) does not comprise measuring the expression of Factor B. In another embodiment, step (b) does not comprise measuring the expression of C5. In an additional embodiment, step (b) does not comprise measuring the expression of CD40.

By “TM peptide” we mean a peptide derived from a 10TM protein, to which the scFv antibody construct of SEQ ID NO: 1 below has specificity (wherein the CDR sequences are indicated by bold, italicised text):

[SEQ ID NO: 1] MAEVQLLESGGGLVQPGGSLRLSCAASGFT

KGLEWV

FTISRDNSKNTLYLQ MNSLRAEDTAVYYCARGTWFDPWGQGTLVTVSSGGGGSGGGGSG GGGSQSVLTQPPSASGTPGQRVTISCS

WYQQL PGTAPKLLIY

GVPDRFSGSKSGTSASLAISGLRSEDE ADYY

FGGGTKLTVLG

Hence, this scFv may be used or any antibody, or antigen binding fragment thereof, that competes with this scFv for binding to the 10TM protein. For example, the antibody, or antigen binding fragment thereof, may comprise the same CDRs as present in SEQ ID NO:1.

It will be appreciated by persons skilled in the art that such an antibody may be produced with an affinity tag (e.g. at the C-terminus) for purification purposes. For example, an affinity tag of SEQ ID NO: 2 below may be utilised:

[SEQ ID NO: 2] DYKDHDGDYKDHDIDYKDDDDKAAAHHHHHH

In one embodiment, presence of pancreatic cancer is identified in the event that the expression in the test sample of IL-3, Integrinα-10, Mucin-1, C1s, GLP-1R, MCP-3, Angiomotin, BTK, CD40 ligand, GM-CSF, IgM, IL-11, IL-16, IL-1-ra, IL-1α, IL-1β, IL-2, IL-7, IL-9, INF-γ, Integrinα-11, JAK3, Leptin, Lewis y, MCP-4, Procathepsin W, PSA, RANTES, Sialyl Lewis x, TM peptide, TNF-α, C4, β-galactosidase, IL-12, TGF-β1, VEGF, IL-8, C3, IFN-γ, IL-10, IL-13, IL-18, IL-6, Lewis x, Eotaxin, C1 esterase inhibitor, MCP-1, TNF-β, GLP-1, IL-5, IL-4, Factor B, C5 and/or CD40 are up-regulated compared to the negative control(s) and/or corresponds to the expression of positive control(s).

In another embodiment, presence of pancreatic cancer is identified in the event that the expression in the test sample of C1q and/or Properdin is down-regulated compared to the negative control(s) and/or corresponds to the expression of positive control(s) Generally, diagnosis is made with an ROC AUC of at least 0.55, for example with an ROC AUC of at least, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99 or with an ROC AUC of 1.00. Preferably, diagnosis is made with an ROC AUC of at least 0.85, and most preferably with an ROC AUC of 1.

Typically, diagnosis is performed using a support vector machine (SVM), such as those available from http://cran.r-project.org/web/packages/e1071/index.html (e.g. e1071 1.5-24). However, any other suitable means may also be used.

Support vector machines (SVMs) are a set of related supervised learning methods used for classification and regression. Given a set of training examples, each marked as belonging to one of two categories, an SVM training algorithm builds a model that predicts whether a new example falls into one category or the other. Intuitively, an SVM model is a representation of the examples as points in space, mapped so that the examples of the separate categories are divided by a clear gap that is as wide as possible. New examples are then mapped into that same space and predicted to belong to a category based on which side of the gap they fall on.

More formally, a support vector machine constructs a hyperplane or set of hyperplanes in a high or infinite dimensional space, which can be used for classification, regression or other tasks. Intuitively, a good separation is achieved by the hyperplane that has the largest distance to the nearest training datapoints of any class (so-called functional margin), since in general the larger the margin the lower the generalization error of the classifier. For more information on SVMs, see for example, Burges, 1998, Data Mining and Knowledge Discovery, 2:121-167.

In one embodiment of the invention, the SVM is ‘trained’ prior to performing the methods of the invention using biomarker profiles from individuals with known disease status (for example, individuals known to have pancreatic cancer, individuals known to have acute inflammatory pancreatitis, individuals known to have chronic pancreatitis or individuals known to be healthy). By running such training samples, the SVM is able to learn what biomarker profiles are associated with pancreatic cancer. Once the training process is complete, the SVM is then able whether or not the biomarker sample tested is from an individual with pancreatic cancer.

However, this training procedure can be by-passed by pre-programming the SVM with the necessary training parameters. For example, diagnoses can be performed according to the known SVM parameters using the SVM algorithm detailed in Table V, based on the measurement of any or all of the biomarkers listed in Table III or Table IV.

It will be appreciated by skilled persons that suitable SVM parameters can be determined for any combination of the biomarkers listed in Table III or Table IV by training an SVM machine with the appropriate selection of data (i.e. biomarker measurements from individuals with known pancreatic cancer status).

Preferably, the method of the invention has an accuracy of at least 60%, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% accuracy.

Preferably, the method of the invention has a sensitivity of at least 60%, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity.

Preferably, the method of the invention has a specificity of at least 60%, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity.

By “accuracy” we mean the proportion of correct outcomes of a method, by “sensitivity” we mean the proportion of all PaC positive sample that are correctly classified as positives, and by “specificity” we mean the proportion of all PaC negative samples that are correctly classified as negatives.

In one embodiment, the individual not afflicted with pancreatic cancer is not afflicted with pancreatic cancer (PaC), chronic pancreatitis (ChP) or acute inflammatory pancreatitis (AIP). More preferably, the healthy individual is not afflicted with any pancreatic disease or condition. Even more preferably, the individual not afflicted with pancreatic cancer is not afflicted with any disease or condition. Most preferably, the individual not afflicted with pancreatic cancer is a healthy individual. By a “healthy individual” we include individuals considered by a skilled person to be physically vigorous and free from physical disease.

However, in another embodiment the individual not afflicted with pancreatic cancer is afflicted with chronic pancreatitis. In still another embodiment, the individual not afflicted with pancreatic cancer is afflicted with acute inflammatory pancreatitis.

As previously mentioned the present method is for determining the presence of pancreatic cancer in an individual. In one embodiment the pancreatic cancer is selected from the group consisting of adenocarcinoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and undifferentiated carcinomas with osteoclast-like giant cells. Preferably, the pancreatic cancer is a pancreatic adenocarcinoma. More preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma, also known as exocrine pancreatic cancer.

In a further embodiment, step (b), (d) and/or step (f) is performed using a first binding agent capable of binding to the one or more biomarkers. It will be appreciated by persons skilled in the art that the first binding agent may comprise or consist of a single species with specificity for one of the protein biomarkers or a plurality of different species, each with specificity for a different protein biomarker.

Suitable binding agents (also referred to as binding molecules) can be selected from a library, based on their ability to bind a given motif, as discussed below.

At least one type of the binding agents, and more typically all of the types, may comprise or consist of an antibody or antigen-binding fragment of the same, or a variant thereof.

Methods for the production and use of antibodies are well known in the art, for example see Antibodies: A Laboratory Manual, 1988, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879693145, Using Antibodies: A Laboratory Manual, 1998, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879695446 and Making and Using Antibodies: A Practical Handbook, 2006, Howard & Kaser, CRC Press, ISBN-13: 978-0849335280 (the disclosures of which are incorporated herein by reference).

Thus, a fragment may contain one or more of the variable heavy (V_(H)) or variable light (V_(L)) domains. For example, the term antibody fragment includes Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_(H) and V_(L) partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544).

The term “antibody variant” includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or constant regions, or other immunointeractive molecule capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.

A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

Molecular libraries such as antibody libraries (Clackson et al, 1991, Nature 352, 624-628; Marks et al, 1991, J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985, Science 228(4705): 1315-7), expressed cDNA libraries (Santi et al (2000) J Mol Biol 296(2): 497-508), libraries on other scaffolds than the antibody framework such as affibodies (Gunneriusson et al, 1999, Appl Environ Microbiol 65(9): 4134-40) or libraries based on aptamers (Kenan et al, 1999, Methods Mol Biol 118, 217-31) may be used as a source from which binding molecules that are specific for a given motif are selected for use in the methods of the invention.

The molecular libraries may be expressed in vivo in prokaryotic (Clackson et al, 1991, op. cit.; Marks et al, 1991, op. cit.) or eukaryotic cells (Kieke et al, 1999, Proc Natl Acad Sci USA, 96(10):5651-6) or may be expressed in vitro without involvement of cells (Hanes & Pluckthun, 1997, Proc Natl Acad Sci USA 94(10):4937-42; He & Taussig, 1997, Nucleic Acids Res 25(24):5132-4; Nemoto et al, 1997, FEBS Lett, 414(2):405-8).

In cases when protein based libraries are used often the genes encoding the libraries of potential binding molecules are packaged in viruses and the potential binding molecule is displayed at the surface of the virus (Clackson et al, 1991, op. cit.; Marks et al, 1991, op. cit; Smith, 1985, op. cit.).

The most commonly used such system today is filamentous bacteriophage displaying antibody fragments at their surfaces, the antibody fragments being expressed as a fusion to the minor coat protein of the bacteriophage (Clackson et al, 1991, op. cit.; Marks et al, 1991, op. cit). However, also other systems for display using other viruses (EP 39578), bacteria (Gunneriusson et al, 1999, op. cit.; Daugherty et al, 1998, Protein Eng 11(9):825-32; Daugherty et al, 1999, Protein Eng 12(7):613-21), and yeast (Shusta et al, 1999, J Mol Biol 292(5):949-56) have been used.

In addition, display systems have been developed utilising linkage of the polypeptide product to its encoding mRNA in so called ribosome display systems (Hanes & Pluckthun, 1997, op. cit.; He & Taussig, 1997, op. cit.; Nemoto et al, 1997, op. cit.), or alternatively linkage of the polypeptide product to the encoding DNA (see U.S. Pat. No. 5,856,090 and WO 98/37186).

When potential binding molecules are selected from libraries one or a few selector peptides having defined motifs are usually employed. Amino acid residues that provide structure, decreasing flexibility in the peptide or charged, polar or hydrophobic side chains allowing interaction with the binding molecule may be used in the design of motifs for selector peptides.

For example:

-   (i) Proline may stabilise a peptide structure as its side chain is     bound both to the alpha carbon as well as the nitrogen; -   (ii) Phenylalanine, tyrosine and tryptophan have aromatic side     chains and are highly hydrophobic, whereas leucine and isoleucine     have aliphatic side chains and are also hydrophobic; -   (iii) Lysine, arginine and histidine have basic side chains and will     be positively charged at neutral pH, whereas aspartate and glutamate     have acidic side chains and will be negatively charged at neutral     pH; -   (iv) Asparagine and glutamine are neutral at neutral pH but contain     a amide group which may participate in hydrogen bonds; -   (v) Serine, threonine and tyrosine side chains contain hydroxyl     groups, which may participate in hydrogen bonds.

Typically, selection of binding agents may involve the use of array technologies and systems to analyse binding to spots corresponding to types of binding molecules.

In one embodiment, the first binding agent(s) is/are immobilised on a surface (e.g. on a multiwell plate or array).

The variable heavy (V_(H)) and variable light (V_(L)) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by “humanisation” of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_(H) and V_(L) partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L) partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” we mean that the said antibodies and F(ab′)₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.

The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications”, J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference.

In one embodiment, the first binding agent immobilised on a surface (e.g. on a multiwell plate or array).

The variable heavy (V_(H)) and variable light (V_(L)) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by “humanisation” of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V_(H) and V_(L) partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L) partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” we mean that the said antibodies and F(ab′)₂ fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.

The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications”, J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference.

Hence, the first binding agent may comprise or consist of an antibody or an antigen-binding fragment thereof. Preferably, the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be selected from the group consisting of: scFv, Fab, and a binding domain of an immunoglobulin molecule.

Preferably, the first binding agent is immobilised on a surface.

The one or more biomarkers in the test sample may be labelled with a detectable moiety.

By a “detectable moiety” we include the meaning that the moiety is one which may be detected and the relative amount and/or location of the moiety (for example, the location on an array) determined.

Suitable detectable moieties are well known in the art.

Thus, the detectable moiety may be a fluorescent and/or luminescent and/or chemiluminescent moiety which, when exposed to specific conditions, may be detected.

For example, a fluorescent moiety may need to be exposed to radiation (i.e. light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected.

Alternatively, the detectable moiety may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualised and/or detected. Examples of suitable enzymes are discussed in more detail below in relation to, for example, ELISA assays.

Alternatively, the detectable moiety may be a radioactive atom which is useful in imaging. Suitable radioactive atoms include ^(99m)Tc and ¹²³I for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as ¹²³I again, ¹³¹I, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, ¹⁷O, gadolinium, manganese or iron. Clearly, the agent to be detected (such as, for example, the one or more biomarkers in the test sample and/or control sample described herein and/or an antibody molecule for use in detecting a selected protein) must have sufficient of the appropriate atomic isotopes in order for the detectable moiety to be readily detectable.

The radio- or other labels may be incorporated into the agents of the invention (i.e. the proteins present in the samples of the methods of the invention and/or the binding agents of the invention) in known ways. For example, if the binding moiety is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as ⁹⁹mTc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can, for example, be attached via cysteine residues in the binding moiety. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate ¹²³I. Reference (“Monoclonal Antibodies in Immunoscintigraphy”, J-F Chatal, CRC Press, 1989) describes other methods in detail. Methods for conjugating other detectable moieties (such as enzymatic, fluorescent, luminescent, chemiluminescent or radioactive moieties) to proteins are well known in the art.

Preferably, the one or more biomarkers in the control sample(s) are labelled with a detectable moiety. The detectable moiety may be selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety. However, it is preferred that the detectable moiety is biotin.

In an additional embodiment step (b), (d) and/or step (f) is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety. Preferably, the second binding agent comprises or consists of an antibody or an antigen-binding fragment thereof. Preferably, the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof. Most preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv, Fab and a binding domain of an immunoglobulin molecule. In one embodiment the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety and an enzymatic moiety. Preferably, the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g. Alexa647).

In one embodiment, the method of the first aspect of the invention comprises or consists of an ELISA (Enzyme Linked Immunosorbent Assay).

Preferred assays for detecting serum or plasma proteins include enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference. Antibody staining of cells on slides may be used in methods well known in cytology laboratory diagnostic tests, as well known to those skilled in the art.

Typically, the assay is an ELISA (Enzyme Linked Immunosorbent Assay) which typically involves the use of enzymes giving a coloured reaction product, usually in solid phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from Escherichia coli provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemi-luminescent systems based on enzymes such as luciferase can also be used.

ELISA methods are well known in the art, for example see The ELISA Guidebook (Methods in Molecular Biology), 2000, Crowther, Humana Press, ISBN-13: 978-0896037281 (the disclosures of which are incorporated by reference).

Conjugation with the vitamin biotin is frequently used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity.

However, step (b), (d) and/or step (f) is alternatively performed using an array. Arrays per se are well known in the art. Typically they are formed of a linear or two-dimensional structure having spaced apart (i.e. discrete) regions (“spots”), each having a finite area, formed on the surface of a solid support. An array can also be a bead structure where each bead can be identified by a molecular code or colour code or identified in a continuous flow. Analysis can also be performed sequentially where the sample is passed over a series of spots each adsorbing the class of molecules from the solution. The solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex, silicon, amongst others), a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, polynucleotides and other suitable molecules and/or conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing a protein molecule, polynucleotide or the like to the solid support. By using well-known techniques, such as contact or non-contact printing, masking or photolithography, the location of each spot can be defined. For reviews see Jenkins, R. E., Pennington, S. R. (2001, Proteomics, 2, 13-29) and Lal et al (2002, Drug Discov Today 15; 7(18 Suppl):S143-9).

Typically the array is a microarray. By “microarray” we include the meaning of an array of regions having a density of discrete regions of at least about 100/cm², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance. The array may also be a macroarray or a nanoarray.

Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

Hence, the array may be the array is a bead-based array or a surface-based array. Preferably, the array is selected from the group consisting of: macroarray, microarray and nanoarray.

In one embodiment, the method according to the first aspect of the invention comprises:

-   -   (i) labelling biomarkers present in the sample with biotin;     -   (ii) contacting the biotin-labelled proteins with an array         comprising a plurality of scFv immobilised at discrete locations         on its surface, the scFv having specificity for one or more of         the proteins in Table III;     -   (iii) contacting the immobilised scFv with a streptavidin         conjugate comprising a fluorescent dye; and     -   (iv) detecting the presence of the dye at discrete locations on         the array surface         wherein the expression of the dye on the array surface is         indicative of the expression of a biomarker from Table III in         the sample.

In an alternative embodiment step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers. Preferably the nucleic acid molecule is a cDNA molecule or an mRNA molecule. Most preferably the nucleic acid molecule is an mRNA molecule.

Hence the expression of the one or more biomarker(s) in step (b), (d) and/or (f) may be performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation. Preferably, the expression of the one or more biomarker(s) in step (b) is determined using a DNA microarray.

In one embodiment, the measuring of the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using one or more binding moieties, each individually capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table III.

In a further embodiment, the one or more binding moieties each comprise or consist of a nucleic acid molecule. Thus, the one or more binding moieties may each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO. However, it is preferred that the one or more binding moieties each comprise or consist of DNA.

Preferably, the one or more binding moieties are 5 to 100 nucleotides in length. More preferably, the one or more nucleic acid molecules are 15 to 35 nucleotides in length. More preferably still, the binding moiety comprises a detectable moiety.

In an additional embodiment, the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); and an enzymatic moiety. Preferably, the detectable moiety comprises or consists of a radioactive atom. The radioactive atom may be selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

However, the detectable moiety of the binding moiety may be a fluorescent moiety (for example an Alexa Fluor dye, e.g. Alexa647).

In one embodiment the sample provided in step (b), (d) and/or (f) is selected from the group consisting of unfractionated blood, plasma, serum, tissue fluid, pancreatic tissue, pancreatic juice, bile and urine. Preferably, the sample provided in step (b), (d) and/or (f) is selected from the group consisting of unfractionated blood, plasma and serum. More preferably, the sample provided in step (b), (d) and/or (f) is plasma. In another preferred embodiment, the sample provided in step (b), (d) and/or (f) is serum.

A second aspect of the present invention provides an array for determining the presence of pancreatic cancer in an individual comprising one or more binding agent as defined in the first aspect of the present invention.

Arrays suitable for use in the methods of the invention are discussed above.

Preferably the one or more binding agents are capable of binding to all of the proteins defined in Table III.

A third aspect of the present invention provides the use of one or more biomarkers selected from the group defined in the first aspect of the invention as a diagnostic marker for determining the presence of pancreatic cancer in an individual. Preferably, all of the proteins defined in Table III are used as diagnostic markers for determining the presence of pancreatic cancer in an individual.

A fourth aspect of the present invention provides a kit for determining the presence of pancreatic cancer comprising:

-   -   A) one or more first binding agent according to the first aspect         of the invention or an array according the second aspect of the         invention; and     -   B) instructions for performing the method according to the first         aspect of the invention.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following tables and figures:

FIGS. 1A-1F: Classification of PaC vs. N

FIG. 1A) Scanned image of an antibody microarray hybridized with a PaC serum. In total, 160 probes, including position markers and controls, were printed in eight 20×8 subarrays per slide. FIG. 1B) Differentially expressed (p<0.05) serum analytes for PaC vs. N. FIG. 10) ROC curve for PaC vs. N based on all antibodies, i.e. using unfiltered data. FIG. 1D) Classification of PaC vs. N, using the SVM prediction values based on all antibodies (red dots-PaC, blue dots-N). The relative expression levels of the top 20 differentially expressed (p<0.02) non-redundant analytes are shown in a heatmap. Red—up-regulated, green—down-regulated, black—equal levels. (FIG. 1E) Validation of scFv antibody specificity, illustrated for a highly differentially expressed (p=0.005) analyte, IL-6, using a 278 human protein array. (FIG. 1F) Validation of scFv antibody specificity, illustrated for a modestly differentially expressed (p=0.04) analyte, IL-10, using a 278 human protein array.

FIGS. 2A-2D: Pre-validation of biomarker signature for PaC vs. N classification

(FIG. 2A) Condensation of the biomarker signature for PaC vs. N classification in the first patient cohort using a LOO procedure combined with a backward elimination strategy. The observed ROC AUC values were plotted against the remaining number of antibodies. (FIG. 2B) The condensed 18-analyte non-redundant biomarker signature obtained from the first patient cohort. (FIG. 2C) The first patient cohort was used as training set, and the output classifier was then tested on a new, independent patient group, the second patient cohort. (FIG. 2D) Pre-validation of the biomarker signature for PaC vs. N classification illustrated by the ROC curve obtained for the classifier on the test set.

FIGS. 3A-3C: Candidate serum biomarker signatures differentiating PaC and pancreatitis

(FIG. 3A) Differentially expressed (p<0.05) serum analytes for PaC vs. ChP, AIP or ChP+AIP+N, respectively. (FIG. 3B) ROC curves for PaC vs ChP, AIP, or ChP+AIP+N classification based on all antibodies, i.e. using unfiltered data. (FIG. 3C) Validation of the antibody microarray data of selected analytes using a 10-plex cytokine sandwich antibody microarray (MSD). Data is only shown for the only analyte, IL-8, for which the majority of the observed signals were above the lower limit of detection for the MSD assay.

FIGS. 4A-4E: Pre-validation of a candidate serum biomarker signature for PaC diagnosis

(FIG. 4A) The first patient cohort, composed of PaC, N, ChP and AIP, was split into a training set (two thirds) and a test set (one third). (FIG. 4B) The condensed 25 non-redundant serum biomarker signature obtained for the training set using a backward elimination strategy. (FIG. 4C) Pre-validation of the condensed 25-analyte biomarker signature for PaC diagnosis, as illustrated by the ROC curve obtained for the classifier on the test set. (FIG. 4D) Performance, expressed as ROC AUC values, of the condensed biomarker signature obtained by the backward elimination strategy (solid line) as compared to that of 25-analyte signatures obtained by either i) 1000 random 25-marker signatures (open circles), ii) lowest p-values (dashed line), or iii) highest fold-change (dotted line). (FIG. 4E) Comparison of the ROC AUC value obtained for the condensed 25-analyte biomarker signature on the test set, when the sample annotation was correct (solid line) or permutated a 1000 times (open circles).

FIG. 5: Schematic outline of the antibody microarray strategy

FIG. 6: ROC-AUC values for differentiation between pancreatic cancer, and normal, chronic pancreatitis, and/or acute inflammatory pancreatitis

ROC-AUC values are shown for marker signatures having all of the Table IV(A) (i.e., core) and Table(B) (i.e., preferred) markers, and increasing numbers of Table(C) (i.e., optional) markers. The best ROC AUC value (0.90) is obtained for a 29 analyte signature, i.e., core markers+preferred markers+15 optional markers. However, all marker combinations had substantial predictive power.

EXAMPLES Materials and Methods Serum Samples

Serum samples were collected at the time of diagnosis, i.e. prior to commencing any therapy, from two independent patient cohorts and stored at −80° C. In the first cohort, serum samples from 103 patients, diagnosed with pancreatic cancer (PaC) (n=34), chronic pancreatitis (ChP) (n=16), autoimmune pancreatitis (AIP) (n=23), or healthy individuals (N) (n=30) (no clinical symptoms) were screened. The patient demographics are described in Table 1. This cohort was randomly split and used as training set and test set. The second cohort, comprised of 45 patients, diagnosed with PaC (n=25), or N (n=20) (for patient demographics, see [14]), was used as an independent test set only, using antibody microarray data recently collected [14]. The size of the sample cohorts was limited by the availability of well-characterized serum samples collected at the time of diagnosis.

Antibody Microarray Analysis

The recombinant antibody microarray analysis was performed using previously in-house optimized protocols [12-15] (see below). Briefly, 121 human recombinant single-chain Fv (scFv) antibodies, targeting 57 mainly immunoregulatory analytes, were used as probes. The specificity, affinity (nM range), and on-chip functionality of the phage-display derived scFvs [16] was ensured by using i) stringent selection protocols [16], ii) multiple clones (4) per target analyte, and iii) a scFv library microarray adapted by molecular design (REF). The planar antibody microarrays (array size; 160×8, <0.5 cm²) were prepared by dispensing the antibodies and controls one-by-one (330 μL/drop) using a non-contact dispenser. The biotinylated serum samples were separately screened and specifically bound analytes were visualized by adding fluorescently labelled streptavidin using a confocal fluorescence scanner. Each individual array data point represents the mean value of four replicates. Chip-to-chip normalization was performed by using a semi-global normalization approach. In accordance to previous studies [12, 15, 17], the correlation coefficient for spot-to-spot reproducibility and array-to-array reproducibility was 0.99 and 0.94, respectively. Selected antibody specificities and microarray data were validated (Table II) using a 234 human protein array and a 10-plex cytokine sandwich antibody microarray, respectively. In addition, several antibody specificities have previously been validated using ELISA, protein arrays, blocking/spiking experiments, and/or mass spectrometry (Table II).

Microarray Data Analysis

The data analysis was performed in R (see below). Briefly, a support vector machine (SVM) was employed to classify the samples as belonging to one of two defined groups (e.g. cancer vs. healthy), using a linear kernel with the cost of constraints set to 1. No attempts were made to tune it in order to avoid the risk of over-fitting. The SVM was trained and tested using a leave-one-out (LOO) cross validation procedure. In two of the comparisons, this training part included the creation of an antibody sub-panel by selecting antibodies that, in the training set, displayed the highest discriminatory power. This selection of antibodies was made using either a direct or a cross-validated backward elimination strategy. Using this approach, condensed candidate biomarker signatures were identified, and subsequently evaluated on independent test sets.

Sensitivity and specificity values were calculated from the SVM decision values, using a threshold level of zero. A receiver operating characteristics (ROC) curve was constructed using the SVM decision values. The area under the curve (AUC) was calculated and used as a measure of prediction performance. Further, the Wilcoxon p-value and the fold change were calculated for each antibody. The candidate biomarker signatures were reported following the recommendations for tumour marker prognostic studies [18].

Serum Samples

After informed consent, serum samples were collected from two independent patient cohorts at the time of diagnosis, i.e. prior to initiation of therapy, and stored at −80° C. PC was verified with histology. Patient cohort 1 was composed of serum samples from 103 patients (Mannheim University Hospital, Germany), diagnosed with pancreatic ductal adenocarcinoma (PaC) (n=34), chronic pancreatitis (hCP) (n=16), autoimmune pancreatitis (AIP) (n=23), or healthy individuals (controls; N) (n=30) (no clinical symptoms). The patient demographics are described in Table 1. This cohort was also randomly split and used as training set (two thirds of the samples) and test set (one third). Patient cohort 2 was composed of 45 patients, diagnosed with PaC (n=25), or N (n=20) (Stockholm South General Hospital and Lund University Hospital, Sweden) (for patient demographics, see [39]), and was adopted using antibody microarray data as recently described [39]. A power analysis (see below) was performed in order to confirm that the size of the sample cohorts was sufficient to provide a statistical power >80%. The main experiments were performed on patient cohort 1, while cohort 2 was used as an independent data set for validation in one experiment (see FIG. 5).

Labelling of Serum Samples

The serum samples were labelled using previously optimized labelling protocols for serum proteomes [39-43]. Briefly, crude serum samples were thawed on ice and 30 μL aliquots were centrifuged (16 000×g for 20 min at 4° C.). Five μL of the supernatant was diluted 45 times in PBS, resulting in a protein concentration of approximately 2 mg/mL. Samples were labelled with EZ-Link® Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill., USA) at a final concentration of 0.6 mM for 2 h on ice with gently vortexing every 20 min. Free biotin was removed by dialysis against PBS for 72 h at 4° C. using 3.5 kDa MW cut-off dialysis units (Thermo Scientific, Rockford, Ill., USA). The samples were aliquoted and stored at −20° C.

Production and Purification of scFv

In total, 121 human recombinant single-chain Fv (scFv) antibody fragments, targeting 57 mainly immunoregulatory biomolecules were selected from the n-CoDeR library [43] and kindly provided by BioInvent International AB, Lund, Sweden, or provided by Prof. Mats Ohlin (Lund University, Sweden) (5 clones against mucin-1). The specificity, affinity (nM range) and on-chip functionality of the phage-display derived scFv was ensured by using i) stringent selection protocols [43], ii) multiple clones (4) per target molecule, and iii) a scFv library microarray adapted by molecular design [44, 45]. The antibody fragments were produced in 100 mL E. coli cultures and purified from either expression supernatants or cell periplasm, using affinity chromatography on Ni-NTA agarose (Qiagen, Hilden, Germany). Bound molecules were eluted with 250 mM imidazole, extensively dialysed against PBS and stored at 4° C. until used for microarray fabrication. The antibody concentration was determined by measuring the absorbance at 280 nm (average 500 μg/mL, range 50-1840 μg/mL).

Fabrication and Processing of Antibody Microarrays

For the production of planar antibody microarrays, we used a set-up previously optimized and validated [39-43, 46]. Briefly, scFvs were arrayed onto black polymer Maxisorb microarray slides (NUNC, Roskilde, Denmark) using a non-contact printer (BioChip Arrayer, PerkinElmer Life & Analytical Sciences, Wellesley, Mass., USA) by depositing approximately 330 μL drops, using piezo technology. Two drops were spotted in each position, allowing the first drop to dry out before the second drop was dispensed. In average, 5 fmol antibody (rang 1.5-25) was deposited per position. In order to ensure adequate statistics and to account for any local defects, each probe was printed in eight replicates. In total, 160 probes, including position markers and control scFvs were printed per slide, oriented in eight 20×8 subarrays. To assist grid alignment during quantification, a row of Alexa647 conjugated Streptavidin (Invitrogen, Carlsband, Calif., USA) (10 μg/mL) was spotted at selected positions. The arrays were blocked in 5% (w/v) fat-free milk powder (Semper AB, Sundbyberg, Sweden) in PBS over night.

The microarray slides were processed in a ProteinArray Workstation (PerkinElmer Life & Analytical Sciences) according to a previously described protocol [42]. Briefly, the arrays were washed with 0.5% (v/v) Tween-20 in PBS (PBS-T) for 4 min at 60 μL/min and then incubated with 75 μL biotinylated serum sample (diluted 1:2, resulting in a total serum dilution of 1:90) in 1% (w/v) fat-free milk powder and 1% (v/v) Tween-20 in PBS (PBS-MT), for 1 h with agitation every 15th second. Next, the arrays were again washed with PBS-T and incubated with 1 μg/mL Alexa-647 conjugated streptavidin in PBS-MT, for 1 h. Finally, the arrays were washed with PBS-T, dried under a stream of nitrogen gas and scanned with a confocal microarray scanner (PerkinElmer Life & Analytical Sciences) at 10 μm resolution, using four different scanner settings of PMT gain and laser power. The intensity of each spot was quantified in the ScanArray Express software v.4.0 (PerkinElmer Life & Analytical Sciences), using the fixed circle method. The local background was subtracted. To compensate for any possible local defects, the two highest and the two lowest replicates were automatically excluded and the mean value of the remaining four replicates was used. For antibodies displaying saturated signals, values from lower scanner settings were scaled and used instead. Chip-to-chip normalization was performed using a semi-global normalization approach previously described [39, 40, 42]. First, the CV for each probe over all samples was calculated and ranked. Second, 15% of the probes that displayed the lowest CV-values over all samples were identified and used to calculate a chip-to-chip normalisation factor for each array. The normalization factor N_(i) was calculated by the formula N_(i)=S_(i)/μ, where S_(i) is the sum of the signal intensities for the antibodies used, averaged over all samples and p is the sample average of S. The intensities were recalculated to log 2 values prior to statistical analysis.

Validation of Antibody Specificity

The specificities of two selected scFvs (anti-IL-6 (2) and anti-IL-10 (1)) were tested using RayBio® 278 Human Protein Array G series (Norcross, Ga., USA), according to protocol provided by the manufacturer. The scFvs were labelled with EZ-Link® Sulfo-NHS-LC-Biotin (Pierce) for 2 h on ice at a 3.5 times molar excess of biotin. Unbound biotin was removed by 72 h dialysis against PBS. In total, 5 μg of antibody was added to each array. Binding was detected using 1 μg/mL Alexa647 conjugated Streptavidin (Invitrogen). PBS was added to one array as a negative control to check for unspecific binding of Streptavidin. The arrays were scanned and the signals from the negative control array were subtracted. In addition, several antibody specificities have previously been validated using well-characterized, standardized serum samples, and independent methods, such as mass spectrometry, ELISA, MSD, and CBA, as well as using spiking and blocking experiments (Table II).

Validation of Array Data

A human Th1/Th2 10-plex MSD (Meso Scale Discovery, Gaithersburg, Md., USA) assay was run in an attempt to validate the antibody microarray results. Each well of the MSD 96-plate had been pre-functionalized with antibodies against IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13 and TNF-α in spatially distinct electrode spots. A total of 34 serum samples (undiluted) were analyzed, including 11 PaC, 11 healthy, 9 ChP and 3 AIP samples (the low number of AIP samples was due to limited sample volumes in that subgroup). The assay was run according to the protocol provided by the manufacturer and the electrochemiluminiscence-based readout was performed in an MSD SECTOR® instrument.

Microarray Data Analysis

All statistics and data analysis was performed in R (http://www.r-project.org). Briefly, a support vector machine (SVM) was employed to classify the samples as belonging to one of two defined groups (e.g. cancer or healthy), using a linear kernel with the cost of constraints set to 1. No attempts were made to tune it in order to avoid the risk of over-fitting. The SVM was trained and tested using a leave-one-out (LOO) cross validation procedure [42]. In two of the comparisons, this training part included creating an antibody sub-panel by selecting antibodies that, in the training set, displayed the highest discriminatory power. This selection of antibodies was made using either a direct or a cross-validated backward elimination strategy. Using this approach, condensed candidate biomarker signatures were identified, and subsequently evaluated on independent test sets.

Sensitivity and specificity values were calculated from the SVM decision values, using a threshold level of zero. A receiver operating characteristics (ROC) curve was constructed using the SVM decision values. The area under the curve (AUC) was calculated and used as a measure of prediction performance. Further, the Wilcoxon p-value and the fold change were calculated for each antibody. The candidate biomarker signatures were reported following the recommendations for tumour marker prognostic studies [47].

Biomarker Signatures Identification

A backward elimination procedure was used for identifying a biomarker signature for distinguishing PaC from healthy individuals. In this approach, one sample at the time was excluded from the dataset. The remaining samples were used for training the SVM by excluding one antibody at the time and performing the classification using the remaining antibodies. When all antibodies had been left out once, the least informative antibody was defined as the one that had been excluded when the smallest Kullback-Leibler (KL) error was obtained for the classification, and was eliminated from the dataset. The LOO procedure was iterated until only one antibody was left and the order by which the antibodies had been eliminated was recorded. The procedure was repeated by excluding a new sample and the iteration continued until all samples had been left out once. A list of the order in which the antibodies were eliminated was generated for each time a sample was excluded. In the end, all samples had been left out once and a consensus list was created where each antibody was assigned a score based on how long it had endured the elimination process, averaged over all iterations performed. Throughout the process, each left out sample was used to test the SVM models built for each new length of antibody subpanels, returning a decision value corresponding to the performance. Consequently, decision values for all samples for any given subpanel length were collected. The corresponding ROC areas were plotted against number of antibodies as a means to evaluate the strength of the data set and the elimination strategy. A condensed signature of 18 analytes was selected from the consensus list and an independent data set from antibody microarray analysis of 25 PaC and 20 N serum samples [39] was used as a test set for pre-validation of the candidate signature. The signature analytes were used in a SVM LOO cross validation procedure in the test set and the ability of the signature to distinguish PaC from N was illustrated in a ROC curve.

A second candidate biomarker signature was generated for classification of PaC among both N, ChP and AIP. First, the data was randomly divided into a training set (two thirds of the samples) and a test set (one third). A modified (even more stringent) backward elimination strategy was used. Instead of leaving out one sample at the time, the SVM was trained only once, using all samples in the training set. Consequently, one elimination list was generated from which a condensed panel of 25 analytes was selected and used to build the SVM in the training set. The model was applied onto the independent test set and a ROC curve was generated. Furthermore, a statistical power analysis was performed to estimate the number of patients required in the test set using the function “power.t.test” in R (decision values assumed normally distributed as suggested by Shapiro-Wilk testing). The observed decision values from the SVM analysis in the training set displayed a standard deviation of 2.87 and a delta value between the groups of 3.47 (difference between mean values). The alpha level (level of significance) was set to 0.05. In addition, the validity of this backward elimination procedure was tested by comparing the performance of the selected signature to 1000 randomly generated signatures of the same length and to signatures generated by selecting the antibodies of the lowest p-value and highest fold-change, respectively. Finally, the strength of the classifier and the data set was tested by comparing the performance of the signature in the test dataset to random data, by generating 1000 permutation of the sample annotations in the test data set.

Results

Classification of PaC Vs. Healthy Controls

In order to identify serum biomarker signature associated with PaC, we performed differential serum protein expression profiling of PaC (n=34) vs. N (n=30), using the first patient cohort. A representative image of an antibody microarray is shown in FIG. 1A, illustrating that dynamic signal intensities, adequate spot morphology and low non-specific background binding were obtained. The results showed that 33 non-redundant protein analytes, including e.g. both Th1 and Th2 cytokines, were found to be differentially expressed (p<0.05), of which all, but the complement proteins C1q and Properdin, were up-regulated in PaC (FIG. 1B).

To investigate whether PaC and N could be differentiated, we ran a SVM LOO cross-validation, based on all antibodies, i.e. using unfiltered data. The data showed that the patient cohorts could be classified with a ROC AUC value of 0.94 (FIG. 10). In FIG. 1D, the samples are plotted by decreasing SVM decision value, and the relative expression pattern of the top 20 differentially expressed analytes (p<0.02) are shown in a heat map. By using a threshold of 0 (default value), the analysis showed that PaC vs. N could be classified with a sensitivity and specificity of 82% and 87%, respectively.

Next, a 278 human protein array was used for validation of selected antibody specificities (FIGS. 1E and 1F). To this end, scFv antibodies against one highly differentially expressed analyte, IL-6 (p=0.005), and one modest differentially expressed analyte, IL-10 (p=0.04), were selected. In both cases, the protein array analysis showed that the scFv antibody fragments bound specifically to their target protein.

Pre-Validation of Condensed Biomarker Signature for PaC Vs. N Classification

In order to test the strength of the classification derived from the first patient cohort (n=64), we first condensed the total number of analytes down to the 18 non-redundant biomarkers contributing the most to the classification, by combining our LOO procedure with an iterative backward elimination strategy. In this process, the Kullback-Leibler divergences error was minimized and used as guide for stepwise removal of the antibodies one-by-one. After each round, the SVM decision values were collected and the corresponding ROC curve and AUC value were calculated. In FIG. 2A, the AUC value is plotted against the number of remaining antibodies, indicating a high and stable classification even when only a few antibodies were included. The 18-analyte condensed candidate serum biomarker signature, composed of a variety of analytes, e.g. cytokines, complement proteins and enzymes, is shown in FIG. 2B. Next, we applied this 18-analyte classifier on a new independent test group, the second patient cohort (n=45) (FIG. 2C). The results showed that the classifier allowed a stratification of patients into PaC vs. N with a ROC AUC value of 0.95 (FIG. 2D), corresponding to a sensitivity of 88% and specificity of 85%. Hence, the data outlined the first pre-validated serum biomarker signature for PaC diagnosis.

Biomarker Signatures Differentiating PaC Vs. Pancreatitis

To test whether cancer could be differentiated from benign conditions in the pancreas, we compared the serum protein expression profile of PaC (n=34) with that of ChP (n=16) or AIP (n=23) using the first patient cohort. In the case of PaC vs. ChP, 15 non-redundant differentially expressed (p<0.05) serum analytes were pin-pointed, of which all but two (IL-4 and IL-12) were up-regulated in PaC (FIG. 3A). Based on unfiltered data, the results showed that PaC and ChP could be differentiated with a ROC AUC value of 0.86 (FIG. 3B), corresponding to a 97% sensitivity and 69% specificity. A total of 49 non-redundant serum analytes were found to be differentially expressed in PaC vs. AIP, with all except for C1q and Properdin, being up-regulated in PaC (FIG. 3A). Again, based on unfiltered data, the results showed that PaC vs. AIP could be classified with a ROC value of 0.99 (FIG. 3B), based on a sensitivity and specificity of 97% and 91%, respectively.

To better reflect the clinical reality, we then investigated whether differences could be deciphered between PaC and the combined, heterogeneous patient group of ChP+AIP+N, using the first patient cohort (n=103). The results showed that 47 non-redundant serum proteins were differentially expressed (p<0.05) (FIG. 3A). A majority of the analytes (45 of 47) were found to be up-regulated in PaC, including a wide range of proteins. Based on unfiltered data, the results showed that PaC could be distinguished from this heterogeneous patient group with a ROC AUC value of 0.85 (FIG. 3B).

In an attempt to validate the array data, an independent 10-plex cytokine sandwich antibody microarray (MSD) was applied (FIG. 3C). However, only 1 of 10 targeted serum analytes, IL-8, was above the lower limit of detection of the MSD assay in a majority of the samples. Still, the observed up-regulation of IL-8 in PaC vs. N, ChP, AIP and combined cohort thereof was statistically confirmed (p<0.05) by the MSD assay in all cases, except for PaC vs. AIP (p=0.29).

Refined Biomarker Signature for PaC Diagnosis

To test the strength of the classification of the entire first patient cohort, including PaC, N, ChP and AIP (n=103), we split the cohort into a training set (two thirds) and test set (one third) (FIG. 4A). Next, a condensed serum biomarker signature composed of the 25 non-redundant analytes contributing the most to the classification in the training set was deciphered by using a direct, iterative backward elimination strategy. The 25-analyte condensed biomarker signature, composed of e.g. cytokines and complement proteins, is shown in FIG. 4B. Next, we applied this 25-analyte classifier on the independent test set (FIG. 4C). The data showed that PaC could be pinpointed with a ROC AUC value of 0.88 (FIG. 4C), outlining a sensitivity and specificity of 73% and 75%, respectively.

To further challenge the classifier, we statistically evaluated its discriminatory power. Firstly, 1000 random signatures of the same length (25 antibodies) were generated in the training set and applied to the test set. The results showed that the AUC values for the random signatures were lower than that of the classifier biomarker signature (AUC=0.88) in 95% of the cases (FIG. 4D). In addition, the AUC values for the corresponding 25-analyte signature selected based on either lowest p-values (AUC=0.77) or highest fold-changes (AUC=0.78) were significantly lower than that of the classifier signature. Hence, the data further indicated the discriminatory power of the classifier, and the applicability of the backward elimination strategy for defining a condensed, high-performing signature. Secondly, the sample annotation of the test set was permutated 1000 times in order to compare the specific classification to random classification of the same number of samples. The results showed that a significantly higher AUC value (0.88 vs. 0.19-0.86, median value of 0.5) was obtained when the correct sample annotation was used than when the random annotation was applied, further demonstrating the strength of the classification.

Discussion

In this study, we have applied affinity proteomics in order to harness the diagnostic power of the immune system to target PaC. We based our approach on the notion that the immune system is exquisitely sensitive to alterations in an individual's state of health, resulting from disease, registering these changes through fluctuations in the levels of, in particular, immunoregulatory analytes. To this end, we designed our antibody microarray to target predominantly these kinds of key regulatory serum analytes. The data showed that PaC-associated candidate biomarkers signatures displaying high diagnostic power could be de-convoluted. In a similar fashion, this affinity proteomic approach has recently allowed the identification of several serological biomarker signatures distinguishing other cancer indications and healthy controls [14-15, 17, 19], further demonstrating the strength of the platform.

We showed for the first time that serum stored information enabling us to discriminate between not only well-defined patient cohorts of PaC vs. controls and PaC vs. pancreatitis, but also between PaC vs. the combined cohort of controls and pancreatitis patients with high confidence. This latter finding was in particular critical, since the candidate biomarker signatures must perform well also in clinical settings where heterogeneous patients groups will be screened.

The clinical impact of a high-performing PaC classifier would be high as no validated serological discriminator is yet in place [2, 7-9, 20-21]. While waiting for a golden classifier to be established, CA-19-9 remains the most useful molecular marker for PaC diagnosis [2, 8-10]. Notably, our data showed a significantly higher median sensitivity (88%) and specificity (85%) for PaC diagnosis than what have been consistently observed for CA-19-9 [2, 9-11], outlining a significant clinically added value. Further, we have recently modelled the impact of new diagnostic possibilities on cost, survival, and quality of life for risk patients, and showed that affinity proteomics had great prospects for becoming a cost-effective tool in screening for PaC (Bolin et al, ms in prep.).

The classifier will perform at its best if early diagnosis, when the tumour is still small and operable, could be performed [2, 9].

In the quest for cancer biomarkers, systemic inflammation is frequently highlighted as a potential confounding factor [23], since cancer development and inflammation has been linked. In early works based on affinity proteomics, the results also often showed that general disease (inflammatory) signatures rather than cancer-specific fingerprints were delineated [24-26]. Notably, we showed here that PaC and pancreatitis could be discriminated with high confidence. Furthermore, the observed signature(s) showed significant differences, i.e. only small overlaps, with those observed for other various inflammatory conditions refs [19, 27] (Carlsson et al., ms in prep.) and other cancers [14-15, 17, 19], further supporting the notion that PaC-specific signatures were deciphered.

The serum immunosignatures could be considered as snapshots of the immune system's activity in a patient at the time of the test. These fingerprints will reflect a combination of direct and indirect (systemic) effects in response to the cancer. Focusing on the cytokine expression profiles, previous reports have shown that pancreatic cancer cell lines expressed a set of cytokines found to be over expressed also in this study, including e.g. IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, and TGF-β1 [28]. Several of these and other cytokines (e.g. VEGF and IL-7) have also been found to be overexpressed in PaC tumour tissue and/or serum/plasma [29-33] further supporting our observations. Although cytokines play a pivotal role in the immune system, interpreting these intricate expression patterns in a biological context is demanding since many of these analytes display pleiotropic functions and PaC is characterized by peculiar cytokine expression patterns [29]. While the expression of e.g. IFN-γ could signal an attempted anti-tumour immune response [29], the immunological environment of PaC has often been found to be in an immunosuppressive site, as illustrated by the concomitant expression of anti-inflammatory cytokines (e.g. TGF-β and IL-10), and potentially inactive proinflammatory cytokines (e.g. IL-12 and IL-18) [29]. A cellular immunosuppression is a striking biological feature of PaC observed in many patients [34]. While Th2 skewed responses have been reported, the Th1/Th2 balance indicated here has also been observed [29, 31, 35]. The cytokine expression pattern has also been found to reflect other parameters, such as survival [14, 29]. Looking at some of the non-cytokine markers, several complements proteins, such as C3, which has been suggested to function in immune surveillance against tumours [36-37], and the carbohydrate antigen Lewis x have also previously been found to be associated with PaC [38].

Taken together, we have addressed a clinical need and demonstrated that immunosignaturing was a powerful approach for deciphering the first pre-validated serological biomarker signatures for PaC diagnosis. This was achieved through a high-performing platform, well-controlled samples and stringent bioinformatic and validation approaches. The potential of the predictor signature will be further validated in follow-up studies, in which independent sample cohorts will be profiled. In the end, these findings will provide novel opportunities for improved PaC diagnosis and thereby enhanced prognosis and clinical management of PaC.

REFERENCES

-   1. Hidalgo, M., Pancreatic cancer. N Engl J Med, 2010. 362(17): p.     1605-17. -   2. Chu, D., W. Kohlmann, and D. G. Adler, Identification and     screening of individuals at increased risk for pancreatic cancer     with emphasis on known environmental and genetic factors and     hereditary syndromes. JOP, 2010. 11(3): p. 203-12. -   3. Jemal A, S. R., Ward E, Hao Y, Xu J, Thun M J., Cancer     statistics, 2009. C A Cancer J Clin, 2009. 59(4). -   4. Pannala, R., et al., New-onset diabetes: a potential clue to the     early diagnosis of pancreatic cancer. Lancet Oncol, 2009. 10(1): p.     88-95. -   5. Warshaw, A. L. and C. Fernandez-del Castillo, Pancreatic     carcinoma. N Engl J Med, 1992. 326(7): p. 455-65. -   6. Galasso, D., A. Carnuccio, and A. Larghi, Pancreatic cancer:     diagnosis and endoscopic staging. Eur Rev Med Pharmacol Sci, 2010.     14(4): p. 375-85. -   7. Chen, R., et al., Proteomics studies of pancreatic cancer.     Proteomics Clin Appl, 2007. 1(12): p. 1582-1591. -   8. Duffy, M. J., et al., Tumor markers in pancreatic cancer: a     European Group on Tumor Markers (EGTM) status report. Ann     Oncol, 2010. 21(3): p. 441-7. -   9. Fry L C, M. K., Malfertheiner P., Molecular markers of pancreatic     cancer: development and clinical relevance. Langenbecks Arch     Surg., 2008. 393(6). -   10. Koopmann J, R. C., Zhang Z, Canto M I, Brown D A, Hunter M, Yeo     C, Chan D W, Breit S N, Goggins M., Serum markers in patients with     resectable pancreatic adenocarcinoma: macrophage inhibitory cytokine     1 versus CA19-9. Clin Cancer Res., 2006. 15(12). -   11. Boeck S, S. P., Holdenrieder S, Wilkowski R, Heinemann V.,     Prognostic and therapeutic significance of carbohydrate antigen 19-9     as tumor marker in patients with pancreatic cancer. Oncology, 2006.     70(4). -   12. Ingvarsson J, L. A., Sjöholm A G, Truedsson L, Jansson B,     Borrebaeck C A, Wingren C., Design of recombinant antibody     microarrays for serum protein profiling: targeting of complement     proteins. J Proteome Res, 2007. 6(9). -   13. Wingren C, I. J., Dexlin L, Szul D, Borrebaeck C A., Design of     recombinant antibody microarrays for complex proteome analysis:     choice of sample labeling-tag and solid support. Proteomics, 2007.     7(17). -   14. Ingvarsson J, W. C., Carlsson A, Ellmark P, Wahren B, Engström     G, Harmenberg U, Krogh M, Peterson C, Borrebaeck C A., Detection of     pancreatic cancer using antibody microarray-based serum protein     profiling. Proteomics, 2008. 8(11). -   15. Carlsson, A., et al., Plasma proteome profiling reveals     biomarker patterns associated with prognosis and therapy selection     in glioblastoma multiforme patients. Proteomics Clinical     Applications, 2010. 4(6-7): p. 591-602. -   16. Söderlind E, S. L., Jirholt P, Kobayashi N, Alexeiva V, Aberg A     M, Nilsson A, Jansson B, Ohlin M, Wingren C, Danielsson L, Carlsson     R, Borrebaeck C A., Recombining germline-derived CDR sequences for     creating diverse single-framework antibody libraries. Nat     Biotechnol., 2000. 18(8). -   17. Carlsson A, W. C., Ingvarsson J, Ellmark P, Baldertorp B, Fernö     M, Olsson H, Borrebaeck C A., Serum proteome profiling of metastatic     breast cancer using recombinant antibody microarrays. Eur J     Cancer, 2008. 44(3). -   18. McShane, L. M., et al., REporting recommendations for tumor     MARKer prognostic studies (REMARK). Nat Clin Pract Oncol, 2005.     2(8): p. 416-22. -   19. Ellmark P, I. J., Carlsson A, Lundin B S, Wingren C, Borrebaeck     C A., Identification of protein expression signatures associated     with Helicobacter pylori infection and gastric adenocarcinoma using     recombinant antibody microarrays. Mol Cell Proteomics., 2006. 5(9). -   20. Garcea, G., et al., Molecular prognostic markers in pancreatic     cancer: a systematic review. Eur J Cancer, 2005. 41(15): p. 2213-36. -   21. Rustgi, A. K., Pancreatic cancer: novel approaches to diagnosis     and therapy. Gastroenterology, 2005. 129(4): p. 1344-7. -   22. Biankin, A. V., et al., Molecular pathogenesis of precursor     lesions of pancreatic ductal adenocarcinoma. Pathology, 2003.     35(1): p. 14-24. -   23. Chechlinska, M., M. Kowalewska, and R. Nowak, Systemic     inflammation as a confounding factor in cancer biomarker discovery     and validation. Nat Rev Cancer, 2010. 10(1): p. 2-3. -   24. Orchekowski, R., et al., Antibody microarray profiling reveals     individual and combined serum proteins associated with pancreatic     cancer. Cancer Res, 2005. 65(23): p. 11193-202. -   25. Gao, W. M., et al., Distinctive serum protein profiles involving     abundant proteins in lung cancer patients based upon antibody     microarray analysis. BMC Cancer, 2005. 5: p. 110. -   26. Miller, J. C., et al., Antibody microarray profiling of human     prostate cancer sera: antibody screening and identification of     potential biomarkers. Proteomics, 2003. 3(1): p. 56-63. -   27. Dexlin-Mellby, L., et al., Tissue proteomic profiling of     preeclamptic placenta tissue using recombinant antibody microarrays.     Proteomics—Clinical Applications, 2010. 4(10-11): p. 794-807. -   28. Bellone, G., et al., Cytokine expression profile in human     pancreatic carcinoma cells and in surgical specimens: implications     for survival. Cancer Immunol Immunother, 2006. 55(6): p. 684-98. -   29. Bellone G, S. C., Mauri F A, Tonel E, Carbone A, Buffolino A,     Dughera L, Robecchi A, Pirisi M, Emanuelli G., Cytokine expression     profile in human pancreatic carcinoma cells and in surgical     specimens: implications for survival. Cancer Immunol     Immunother., 2006. 55(6). -   30. Chang S T, Z. J., Horecka J, Kunz P L, Ford J M, Fisher G A, Le     Q T, Chang D T, Ji H, Koong A C., Identification of a biomarker     panel using a multiplexed proximity ligation assay improves accuracy     of pancreatic cancer diagnosis. J Transl Med., 2009. 7(105). -   31. Poch B, L. E., Ramadani M, Gansauge S, Beger H G, Gansauge F.,     Systemic immune dysfunction in pancreatic cancer patients.     Langenbecks Arch Surg; 2007. 392(3). -   32. Wigmore S J, F. K., Sangster K, Maingay J P, Garden O J, Ross J     A., Cytokine regulation of constitutive production of interleukin-8     and -6 by human pancreatic cancer cell lines and serum cytokine     concentrations in patients with pancreatic cancer. Int J     Oncol., 2002. 21(4). -   33. Frick V O, R. C., Wagner M, Graeber S, Grimm H, Kopp B, Rau B M,     Schilling M K., Enhanced ENA-78 and IL-8 expression in patients with     malignant pancreatic diseases. Pancreatology., 2008. 8(4-5). -   34. Ungefroren, H., et al., Immunological escape mechanisms in     pancreatic carcinoma. Ann N Y Acad Sci, 1999. 880: p. 243-51. -   35. Schmitz-Winnenthal F H, V. C., Z′graggen K, Galindo L, Nummer D,     Ziouta Y, Bucur M, Weitz J, Schirrmacher V, Büchler M W, Beckhove     P., High frequencies of functional tumor-reactive T cells in bone     marrow and blood of pancreatic cancer patients. Cancer Res., 2005.     65(21). -   36. Chen R, P. S., Cooke K, Moyes K W, Bronner M P, Goodlett D R,     Aebersold R, Brentnall T A., Comparison of pancreas juice proteins     from cancer versus pancreatitis using quantitative proteomic     analysis. Pancreas, 2007. 34(1). -   37. Yu K H, R. A., Blair I A., Characterization of proteins in human     pancreatic cancer serum using differential gel electrophoresis and     tandem mass spectrometry. J Proteome Res., 2005. 4(5). -   38. Nakano M, N. T., Ito T, Kitada T, Hijioka T, Kasahara A, Tajiri     M, Wada Y, Taniguchi N, Miyoshi E., Site-specific analysis of     N-glycans on haptoglobin in sera of patients with pancreatic cancer:     a novel approach for the development of tumor markers. Int J     Cancer., 2008. 122(10). -   39. Ingvarsson J, Wingren C, Carlsson A, et al. Detection of     pancreatic cancer using antibody microarray-based serum protein     profiling. Proteomics 2008; 8. -   40. Carlsson A, Wingren C, Ingvarsson J, et al. Serum proteome     profiling of metastatic breast cancer using recombinant antibody     microarrays. Eur J Cancer 2008; 44: 472-80. -   41. Wingren C, Ingvarsson J, Dexlin L, Szul D, Borrebaeck C A K.     Design of recombinant antibody microarrays for complex proteome     analysis: choice of sample labeling-tag and solid support.     Proteomics 2007; 7:3055-65. -   42. Carlsson A, Persson O, Ingvarsson J, et al. Plasma proteome     profiling reveals biomarker patterns associated with prognosis and     therapy selection in glioblastoma multiforme patients. Proteomics     Clin Appl 2010; 4:1-12. -   43. Söderlind E, Strandberg L, Jirholt P, et al. Recombining     germline-derived CDR sequences for creating diverse single-framework     antibody libraries. Nat Biotechnol 2000; 18:852-6. -   44. Borrebaeck C A K, Wingren C. Design of high-density antibody     microarrays for disease proteomics: key technological issues. J     Proteomics 2009; 72:928-35. -   45. Borrebaeck C A K, Wingren C. High-throughput proteomics using     antibody microarrays: an update. Expert Rev Mol Diagn 2007; 7:     673-86. -   46. Ellmark P; ingvarsson J, Carlsson A, Lundin B S, Wingren C,     Borrebaeck C A K. Identification of protein expression signatures     associated with Helicobacter pylori infection and gastric     adenocarcinoma using recombinant antibody microarrays. Mol Cell     Proteomics 2006; 5:1638-46. -   47. McShane L M, Altman D G, Sauerbrei W, Taube S E, Gion M, Clark     G M. REporting recommendations for tumor MARKer prognostic studies     (REMARK). Nat Clin Pract Oncol 2005; 2:416-22.

TABLE I Patient demographics of the first patient cohort No. of Gender Mean Age Class patients (M/F/unknown) (SD) Range PaC  34 18/12/4 65.0 42-93 (10.4) N  30 15/15/0 33.2 24-53 (8.6) ChP  16 12/4/0  48.8 32-73 (14.2) AIP  23 11/11/1 42.4 14-74 (18.3) All 103 56/42/5 48.2 14-93 (18.1)

TABLE II Summary of serum biomarkers analyzed by the antibody microarrays Antigen (no. of Antigen clones) (no. of clones) Angiomotin (2) IL-2 (3) β-galactosidase (1) IL-3 (3) Bruton tyrosine kinase IL-4 (4)** BTK (1) IL-5 (3)** C1 Esterase inhibitor IL-6 (4)*/** (1) IL-7 (2) C1q (1)** IL-8 (3)** C1s (1) IL-9 (3) C3 (2)** Integrin α10 (1) C4 (1)** Integrin α11 (1) C5 (2)** Leptin (1) CD40 (4) Lewis x (2) CD40 ligand (1) Lewis y (1) CT-17 (control) (1) MCP-1 (3)** Digoxin (control) (1) MCP-3 (1) Eotaxin (3) MCP-4 (2) Factor B (1)** Mucin-1 (6) GLP-1 (1) Procathepsin W (1) GLP-1 R (1) Properdin (1)** GM-CSF (3) PSA (1) IFN-γ (2) RANTES (2) IgM (1) Sialyl Lewis x (1) IL-10 (3)* TGF-β1 (3) IL-11 (3) TM peptide (1) IL-12 (4)** TNF-α (2) IL-13 (2)** TNF-β (4)** IL-16 (2) Tyrosine protein kinase IL-18 (3) JAK3 (1) IL-1α (3)** VEGF (4)** IL-1β (3) IL-1-ra (3) *Antibody specificity determined by protein array. **Antibody specificity previously validated by ELISA, protein array, blocking/spiking experiments, and/or mass spectrometry.

TABLE III Pancreatic Cancer Diagnostic Biomarkers Biomarker name Exemplary sequences Interleukin-7 (IL-7) AK226000, AB102893, AB102885, P13232 Integrin α-10 Hs158237; 075578 B-galactosidase P16278 Bruton's tyrosine kinase (BTK) Q06187 Complement protein C1q (C1q) IPR001073, PR00007 Complement protein C1s (C1s) P09871 B cell receptor μ □ chain (IgM) e.g. P01871 (not complete protein); isotype-specific for IgM on Ramos B cells¹⁾ Interleukin-9 (IL-9) P15248 Integrin α-11 Q9UKX5 Janus kinase 3 protein tyrosine P52333 kinase (JAK3) Procathepsin W P56202 Properdin P27918 TM peptide (10TM protein) NA-see above Tumour necrosis factor-α (TNF-α) P01375 Angiomotin AAG01851; Q4VCS5 Complement-1 esterase inhibitor P05155 (C1-INH) Complement protein C3 (C3) BC150179, BC150200; P01024 Complement protein C4 (C4) BC151204, BC146673, AY379959, AL645922, AY379927, AY379926, AY379925 Complement protein C5 (C5) BC113738, BC113740, DQ400449, AB209031, P01031 CD40 Q6P2H9 Eotaxin P51671 Complement Factor B (Factor B) P00751 Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 receptor P43220 (GLP-1 R) Granulocyte-macrophage colony- P04141 stimulating factor (GM-CSF) Interleukin-10 (IL-10) P22301 Interleukin-11 (IL-11) P20809 Interleukin-12 (IL-12) O60595 Interleukin-13 (IL-13) P35225 Interleukin-18 (IL-18) Q14116 Interleukin-1α (IL-1α) P01583 Interleukin-1β (IL-1β) P01584 Interleukin-2 (IL-2) P60568 Interleukin-3 (IL-3) P08700 Interleukin-4 (IL-4) P05112 Interleukin-5 (IL-5) BC066282, CH471062, P05113 Interleukin-6 (IL-6) P05231 Interleukin-8 (IL-8) CR623827, CR623683, DQ893727, DQ890564, P10145 Interferon-γ (INF-γ) P01579 Leptin P41159 Lewis X/CD15 Carbohydrate structure (not applicable) Lewis y Carbohydrate structure (not applicable) Monocyte chemotactic protein-1 P13500 (MCP-1) Mucin-1 P15941 Prostate specific antigen (PSA) P07288 RANTES P13501 Sialyl Lewis x Carbohydrate structure (not applicable) Transforming growth factor-1 P01137 (TGF-b1) Tumour necrosis factor-β (TNF-β) P01374 Vascular endothelial growth factor P15692, P49765, P49767, =O43915 (VEGF) CD40 ligand P29965 Interleukin-16 (IL-16) Q05BE6, Q8IUU6, B5TY35 Interleukin-1ra (IL-1ra) P18510 Monocyte chemotactic protein-3 BC112258, BC112260, (MCP-3) BC092436, BC070240 Monocyte chemotactic protein-4 Q99616 (MCP-4)

TABLE IV Pancreatic Cancer Diagnostic Biomarkers (A) Core biomarkers Biomarker name Interleukin-7 (IL-7) Integrin α-10 (B) Preferred biomarkers Biomarker name B-galactosidase Bruton's tyrosine kinase (BTK) Complement protein C1q (C1q) Complement protein C1s (C1s) B cell receptor μ □ chain (IgM) Interleukin-9 (IL-9) Integrin α-11 Janus kinase 3 protein tyrosine kinase (JAK3) Procathepsin W Properdin TM peptide Tumour necrosis factor-α (TNF-α) (C) Optional additional biomarkers Biomarker name Angiomotin Complement-1 esteras inhibitor (C1-INH) Complement protein C3 (C3) Complement protein C4 (C4) Complement protein C5 (C5) CD40 Eotaxin Complement Factor B (Factor B) Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 receptor (GLP-1 R) Granulocyte-macrophage colony-stimulating factor (GM-CSF) Interleukin-10 (IL-10) Interleukin-11 (IL-11) Interleukin-12 (IL-12) Interleukin-13 (IL-13) Interleukin-18 (IL-18) Interleukin-1α (IL-1α) Interleukin-1β (IL-1β) Interleukin-2 (IL-2) Interleukin-3 (IL-3) Interleukin-4 (IL-4) Interleukin-5 (IL-5) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interferon-γ (IFN-γ) Leptin Lewis X/CD15 Lewis y Biomarker name Monocyte chemotactic protein-1 (MCP-1) Mucin-1 Prostate specific antigen (PSA) Rantes Sialyl Lewis x Transformign growth factor-1 (TGF-b1) Tumour necrosis factor-β (TNF-β) Vascular endothelial growth factor (VEGF) CD40 ligand Interleukin-16 (IL-16) Interleukin-1ra (IL-1ra) Monocyte chemotactic protein-3 (MCP-3) Monocyte chemotactic protein-4 (MCP-4)

TABLE V Pancreatic Cancer Diagnostic Biomarker Subsets PaC vs N + PaC vs N Chp + AIP PaC vs ChP PaC vs AIP Biomarker name P value Backward P value backward P value P value A CD40 X X X X X Interleukin-12 (IL-12) X X X X X X Interleukin-3 (IL-3) X X X X X X Interleukin-4 (IL-4) X X X X X Interleukin-8 (IL-8) X X X X Monocyte chemotactic X X X X X protein-1 (MCP-1) Mucin-1 X X X X X Transforming growth X X X X X X factor, beta-1 (TGF-b1) Tumour necrosis X X X X X factor-β (TNF-β) Vascular endothelial X X X X X X growth factor (VEGF) B B-galactosidase X X X Bruton's tyrosine kinase (BTK) X X X X CD40 ligand X X X X Complement protein C1q (C1q) X X X Complement protein C3 (C3) X X X X Glucagon-like peptide-1 (GLP-1) X X X B cell receptor μ □chain (IgM) X X X X X Interleukin-10 (IL-10) X X X Interleukin-11 (IL-11) X X X X Interleukin-13 (IL-13) X X X Interleukin-16 (IL-16) X X X X X Interleukin-18 (IL-18) X X X X Interleukin-1α (IL-1α) X X X X X Interleukin-1ra (IL-1ra) X X X X Interleukin-5 (IL-5) X X X X Interleukin-6 (IL-6) X X X X Interleukin-7 (IL-7) X X X X X Interferon-γ (INF-γ) X X X X Integrin α-11 X X X Janus kinase 3 protein X X X tyrosine kinase (JAK3) Lewis x/CD15 X X X Procathepsin W X X X Properdin X X X X X Sialyl Lewis x X X X C Complement protein C1s (C1s) X X X Eotaxin X X X X Glucagon-like peptide-1 X X X receptor (GLP-1 R) Integrin α-10 X X X X Monocyte chemotactic X X X X protein-3 (MCP-3) D Complement-1 esteras X X inhibitor (C1-INH) Complement protein C5 (C5) X X X X Tumour necrosis factor-α X X X X (TNF-α) E Interleukin-9 (IL-9) X X F Granulocyte-macrophage colony- X X stimulating factor (GM-CSF) Interleukin-2 (IL-2) X X X Leptin X X Lewis y X X Prostate specific antigen (PSA) X X Rantes X X G Angiomotin X Complement protein C4 (C4) X Complement Factor B (Factor B) X H Interleukin-1β (IL-1β) X Monocyte chemotactic X protein-4 (MCP-4) TM peptide X

TABLE VI Trained SVM program The following parameters were obtained using the e1071 1.5-24 SVM, available from cran.r-project.org/web/packages/e1071/index.html. (A) - Definition of a condensed biomarker signature for PaC vs all ( N+Chp +AIP) using a backward elimination strategy filnamn <- “PaC vs all training set.txt” group1 <- “other” group2 <- “PaC” # Include source(“NaiveBayesian”) library(e1071) # Hamta data rawfile <- read.delim(filnamn) # Las in grupper groups <- rawfile[,2] # Hamta provnamn i datafilen samplenames <- as.character(rawfile[,1]) # Skapa dataset ur rafilen data <- t(rawfile[,−c(1,2)]) # Log # data <- log(data)/log(2) # antal prover nsamples <- ncol(data) # Skapa antikroppsnamnlista ur NYA datafilen ProteinNames <- read.delim(filnamn,header=FALSE) ProteinNames <- as.character(as.matrix(ProteinNames)[1,]) ProteinNames <- ProteinNames[−(1:2)] # Kolla antal Ab i nya datasetet antal <- length(ProteinNames) # Ge rätt prov- och Ab-namn rownames(data) <- ProteinNames colnames(data) <- samplenames # Skapa subsets subset1 <- is.element(groups , strsplit(group1,“,”)[[1]]) subset2 <- is.element(groups , strsplit(group2,“,”)[[1]]) # Skapa factorlista svmfac <- factor(rep(‘rest’,ncol(data )),levels=c(group1,group2,‘rest’)) svmfac[subset1] <- group1 svmfac[subset2] <- group2 svmfac <- svmfac[subset1|subset2] # Skapa vektor för K-L felen där det minsta för varje signaturlängd sparas smallestErrorPerLength <- rep(NA,antal) # Beräkna medelvärde för varje Ab över alla prov som är med averages <- apply(data, 1, mean) # Skapa vektor för Ab-ordningen efter K-L felen som erhållits när # respektive antikropp var satt till medelvärde. abOrder <- rep(NA,antal) # Skapa ett dataset att eliminera i elimData <- data[,subset1|subset2] # Lista att förvara SVM-modellerna i models <- numeric(nsamples) # Skapa variabel för att hålla reda på hur många Ab som tagits bort borttagna <- 0 #################################################################### # BEGIN BACKELIM ################################################ #################################################################### print(Sys.time( )) # Kör tills bara två analyter återstår for (j in 1:(antal-1)) {  # Check if groups are given in correct order  control < - as.numeric(svmfac)  if(sum(control[subset1]) > sum(control[subset2]))  {   print(“ERROR: Change order of your groupl and group2!!!”)   break  }  # För varje signaturlängd, där alla är med från början, träna en modell för  # varje N-1 kombiantion av prover med den data som finns i elimData  for (i in 1:nsamples)  {   # Modellerna sparas i en array av listor kallad models   models[i] <- list(svm(t(elimData[,−i]), svmfac[−i], kernel=“linear”))  }  # Nu är alla modeller som behövs för LOO tränade och ska testas på elimData.  # I elimData sätts forst en analyt till medelvärde, sen testas var och en av  # modellerna med det prov som var borttaget när den tranades.  # När alla modellerna är testade en gang beraknas KL-fel som sparas i errors.  # Nu sätts nasta analyt till medelvärde och testprocessen görs om, tills alla  # analyter varit medelvardeseliminerade en gång. Resultatet blir en KL-fel  # lista lika lang som antalet analyter som är kvar i datasetet.  # Skapa en lista med K-L fel en viss signaturlangd (antal + 1 − j lång)  # där areorna för varje körning där en Ab i taget har satts till medelvärde  errors <- testModels (models, elimData, averages)  # Lägg namnet på Ab med sämst inverkan på felet i abOrder  abOrder[j] <- getWorstAb(errors, row.names(elimData))  # Lägger till värdet på det minsta felet  smallestErrorPerLength[j] <- getSmallestError(errors)  # Tar bort sämsta Ab ur medelvärdeslistan  averages <- getNewAverages(errors, averages)  # Tar bort sämsta Ab ur elimData  elimData <- getNewElimData(errors, elimData)  # Noterar att en Ab tagits bort  borttagna <- borttagna + 1  # Ange hur många analyter som eliminerats, samt vad klockan är.  print(paste(j, “analytes eliminated @”, Sys.time( )), sep=“ ”) } # Lägg till namnet på sista analyetn, som aldrig blen eliminerad abOrder[length(abOrder)] <- setdiff(ProteinNames, abOrder) # Spara resultatet till fil filename <- paste(“Backward elimination result(“,rnorm(1)+1,”).txt”,sep=“ ”) write.table(cbind(smallestErrorPerLength,abOrder), file=filename, sep=“\t”, quote = F,row.names = F) #################################################################### # FUNCTIONS ################################################ #################################################################### # getWorstAb: Rapporterar namnet på antikroppen som kommer tas bort # (den där ROC-arean var som störst) getWorstAb <- function(errors, abNames) {  return(abNames[order(errors, decreasing = F)[1]]) } # testModels: testar alla modeller som finns i ‘models’ med alla # analyser satta till medelvärde en gång testModels <- function(models, elimData, averages) {  nsamples <- ncol(elimData)  d <- as.numeric(svmfac)-1  y <- numeric(nsamples)  E <- numeric(nsamples)  analytes <- nrow(elimData)  errors <- numeric(nrow(elimData))  for(k in 1:analytes)  {   # Sätt analyt k till medelvärde i elimData    # Men spara först analytens orginalvärde   backup <- elimData[k,]   elimData[k,] <- averages[k]   # Gör LOO loop för datasetet med de redan färdiga modellena   for (i in 1:nsamples)   {    pred <- predict(models[[i]] , t(elimData[,i]), decision.values=TRUE)    #spara decision values    y[i] <- as.numeric(attributes(pred)$decision.values)   }   # Beräkna “sannolikheterna”   y = 1−(1/(1 + exp(−y)))   # Beräkna KL-fel när aktuell analyt är eliminerad   for (i in 1:nsamples)   {    E[i] <- -(d[i]*log(y[i])+(1−d[i])*log(1−y[i]))   }   # Spara felet   errors[k] <- sum(E)   # Lägg tillbaka analyten   elimData[k,] <- backup  }  return( errors ) } # getNewElimData: Väljer vilken antikropp som ska tas bort ur tränigsdatan och tar bort den getNewElimData <- function(errors, elimData) {  # Positionen för det minsta felet  tasBort <- order(errors,decreasing = F)[1]  return(elimData[−tasBort,]) } # getSmallestError: Rapporterar minsta K-L felet getSmallestError <- function(errors) {  return(min(errors)) } # getNewAverages: skapar en ny lista med medelvärden efter att en analyt # eliminerats. getNewAverages <- function(errors, averages) {  # Positionen för det minsta felet  tasBort <- order(errors, decreasing = F)[1]  return(averages[−tasBort]) } # getRemovedAb: tar fram ID på analyt som eliminerats getRemovedAb <- function(errors, abNames) {  return(abNames[order(errors, decreasing = T) [1]]) } (B) - Definition of a condensed biomarker signature for PaC vs N using a modified backward elimination strategy # Datafil och grupper   filnamn <- “PaC_vs_N_dataset.txt”   group1 <- “N”   group2 <- “PaC”   # Läs in datafil   rawfileORG <- read.delim(filnamn)   # Läs in grupper   groupsORG <- rawfileORG[,2]   # Läs in data   dataORG <- log(t(rawfileORG[,-c(1,2)]))   # Läs in Ab-namn   ProteinNames <- read.delim(filnamn,header=FALSE)   ProteinNames <- as.character(as.matrix(ProteinNames)[1,])   ProteinNames <- ProteinNames[−(1:2)]   # Kalla Ab rätt namn   rownames(dataORG) <- ProteinNames   # Kalla prover rätt namn   samplenamesORG <- as.character(rawfileORG[,1])   colnames(dataORG) <- samplenamesORG   # Kontrollera antalet prover   NoSamples <- dim(rawfileORG)[1]   # Kontrollera antalet Ab   NoAntibodies <- dim(rawfileORG)[2] - 2   # Skapa subsets utifrån grupepr   subsetORG1 <- is.element(groupsORG , strsplit(group1,“,”)[[1]])   subsetORG2 <- is.element(groupsORG , strsplit(group2,“,”)[[1]])   # Skapa faktorer utifrån subsets   svmfacORG <- factor(rep(‘rest’,ncol(dataORG   )),levels=c(group1,group2,'rest'))   svmfacORG[subsetORG1] <- group1   svmfacORG[subsetORG2] <- group2   # Skapa vektor och array får ROC-areor respektive Signaturer   # från varje körning utan A provet   BestROCsForEachRun <- rep(NA,NoSamples*NoAntibodies)   dim(BestROCsForEachRun) <- c(NoSamples,NoAntibodies)   AbRemovalOrderForEachRun <- rep(NA,NoSamples*NoAntibodies)   dim(AbRemovalOrderForEachRun) <- c(NoSamples,NoAntibodies) # För varje prov i datasetet: for(A in 1:NoSamples) # for(A in NoSamples:1) {  # Hämta data från orginal-råfilen för alla prover utom A  rawfile<-rawfileORG[−A,]  # Hämta provnamn i NYA datafilen  samplenames <- as.character(rawfile[,1])  # Hämta grupper i NYA datafilen  groups <- rawfile[,2]  # Skapa dataset ur NYA datasete  runData <- t(rawfile[,−c(1,2)])  # Skapa antikroppsnamnlista ur NYA datafilen  ProteinNames <- read.delim(filnamn,header=FALSE)  ProteinNames <- as.character(as.matrix(ProteinNames)[1,])  ProteinNames <- ProteinNames[−(1:2)]  # Kolla antal Ab i nya datasetet  antal <- length(ProteinNames)  # Ge rätt prov- och Ab-namn  rownames(runData) <- ProteinNames  colnames(runData) <- samplenames  # Skapa nya subsets  subset1 <- is.element(groups , strsplit(group1,“,”)[[1]])  subset2 <- is.element(groups , strsplit(group2,“,”)[[1]])  # Skapa fly factorlista  svmfac <- factor(rep(‘rest’,ncol(runData )),levels=c(group1,group2,‘rest’)) svmfac[subset1] <- group1 svmfac[subset2] <- group2  # Skapa vektor för ROC-areor där den bästa för varje signaturlängd sparas  bestRocPerLength <- rep(NA,antal)  # Beräkna medelvärde för varje Ab över alla prov som är med  averages <- apply(runData, 1, mean)  # Skapa vektor för Ab-ordningen efter ROC-areorna som erhållits när  # respektive antikropp var satt till medelvärde.  abOrder<-rep(NA,antal)  # Skapa tränings och testset att köra  trainData <- runData  testData <- runData  # Skapa variabel för att hålla reda på hur många Ab som tagits bort  borttagna <- 0  # Kör lika många gånger som antalet Ab - 1  for(j in 1: (antal-1)  {   # Skapa en lista med ROC-areor en viss signaturlängd (antal + 1 − j lang)   # där areorna för varje körning där en Ab i taget har satts till medelvärde   ROClist <- svmForAbList(antal-borttagna, trainData, svmfac, averages)   # Lägg Ab med sämst inverkan på ROC-area   abOrder[j] <- getRemovedAb(ROClist,row.names(trainData))   # Skapa fly träningsdata där sämsta Ab tas bort   trainData <- getNewTrainData(ROClist, trainData)   # Tar bort sämsta Ab ur medelvärdeslistan   averages <- getNewAverages(ROClist, averages)   # Noterar att en Ab tagits bort   borttagna <- borttagna + 1   # Lägger till värdet på den bästa ROC-arean   bestRocPerLength[j]<-getBestROC(ROClist)  }  # Lägg till den bästa arean för aktuell längd i en lista  BestROCsForEachRun[A,] <- bestRocPerLength  # Lägg till vilken Ab som togs bort för aktuell längd i en lista  AbRemovalOrderForEachRun[A,] <- abOrder  # Skriv vilken körning som genomförts till prompten  print(paste(j, “in”, A,“of”, NoSamples,“at”,Sys.time( ))) { # Include source(“NaiveBayesian”) library(e1071) # Skapar en listamed ROC-areor för en vända med ett antal antikroppar # där alla antikroppar satts till medelvärde en gång svmForAbList <- function(abNumber, trainData, svmfac, averages) {  testData <- trainData  ROClist <- rep(NA,abNumber)  for(k in 1:abNumber) # Byter en variabel i traningsdata till medelvärden,  {          # kör svmloo, byter tillbaka till orginalvärdena.   testData[k,] <- averages[k]   #ROClist[k] <- svmLOOvaluesBE(trainData, testData, svmfac)   ROClist[k] <- svmLOOvaluesProb(trainData, testData, svmfac)   testData[k,] <- trainData[k,]  }  return(ROClist) } # Rapporterar namnet på antikroppen som kommer tas bort # (den där ROC-arean var som storst) getRemovedAb <- function(ROClist, abNames) {  return(abNames[order(ROClist, decreasing = T)[1]]) } # Rapporterar största ROC-arean getBestROC <- function(ROClist) {  return(max(ROClist)) } # Väljer vilken antikropp som ska tas bort ur tränigsdatan och tar bort den getNewTrainData <- function(ROClist, trainData) {  # Positionen för den största ROC-arean  tasBort <- order(ROClist,decreasing=T)[1]  return(trainData[−tasBort,]) } # Väljer vilken antikropp som ska tas bort ur averages getNewAverages <- function(ROClist, averages) {  # Positionen för den största ROC-arean  tasBort <- order(ROClist,decreasing=T)[1]  return(averages[−tasBort]) } svmLOOvaluesBE <- function(trainData, testData, svmfac) {  nsamples <- ncol(trainData)  res <- numeric(nsamples)  sign <- numeric(nsamples)  for (i in 1:nsamples)   {   svmtrain <- svm(t(trainData[,−i]) , svmfac[−i] , kernel=“linear” )   pred <- predict(svmtrain , t(testData[,i]) , decision.values=TRUE)   res[i] <- as.numeric(attributes(pred)$decision.values)   fcn <- colnames(attributes(pred)$decision.values)[1] if(fcn==paste(levels(svmfac)[1],“/”,levels(svmfac)[2],sep=“ ”)){sign[i]<- 1} if(fcn==paste(levels(svmfac)[2],“/”,levels(svmfac)[1],sep=“ ”)){sign[i]<- −1}  }  res <- sign * res  ROCdata <- myROC(res,svmfac)  return(ROCdata[1]) } # Tränar svm med viss signatur på viss data och testar på ett prov test1sample <- function(dataORG, svmfacORG, A, signatureAbs) {  svmtrain <- svm(t(dataORG[signatureAbs,−A]) , svmfacORG[−A] , kernel=“linear”)  pred <- predict(svmtrain , t(dataORG[signatureAbs,A]) , decision.values=TRUE)  res <- as.numeric(attributes(pred)$decision.values)  return (res) } svmLOOvaluesProb <- function(trainData, testData, svmfac) {  nsamples <- ncol(trainData)  #res <- numeric(nsamples)  #sign <- numeric(nsamples)  d <- as.numeric(svmfac)−1  y <- numeric(nsamples)  E <- numeric(nsamples)  for (i in 1:nsamples)  {   svmtrain <- svm(t(trainData[,−i]) , svmfac[−i] , kernel=“linear”)   pred <- predict(svmtrain , t(testData[,i]), decision.values=TRUE)   y[i] <- as.numeric(attributes(pred)$decision.values)  }   y = 1−(1/(1 + exp(−y)))    for (i in 1:nsamples)    {     E[i] <- -(d[i]*log(y[i])+(1−d[i])*log(1−y[i]))    }    return(1/sum(E)) } (C) -Test of signature defined in Table 5(A) aprk-c( ″ IL-3 (1) ″, ″ C3 (1) ″, ″ C5 (1) ″, ″ IL-7 (2) ″, ″ IL-4 (3) ″, ″ CD40 (2) ″, ″ TGF-131 (1) ″, ″ IL-12 (1) ″, ″ GM-CSF (1) ″, ″ Properdin ″, ″ IgM (B) ″, ″ VEGF (3) ″, ″ IL-16 (1) ″, ″ MUC-1 (P3-15) ″, ″ IL-1a (1) ″, ″ TNF-b (1) ″, ″ Integrina-10 ″, ″ Cl est. inh. ″, ″ MCP-1 (3) ″, ″ MCP-3 (2) ″, ″ IL-2 (3) ″, ″ Eotaxin (3) ″, ″ IL-11 (2) ″, ″ TNF-a (1) ″, ″ IL-1-ra (3) ″) library (MASS) library (gplots) library (e1071) source(“C:/Program/R/R-2.8.1/library/NaiveBayesian”) filnamn<-“PaC all data.txt” rawfile <- read.delim(filnamn) samplenames <- as.character(rawfile[,1]) groups <- rawfile[,2] data <- t(rawfile[,−c(1,2)]) ProteinNames <- read.delim(filnamn,header=FALSE) ProteinNames <- as.character(as.matrix(ProteinNames)[1,]) ProteinNames <- ProteinNames[−(1:2)] rownames(data) <- ProteinNames colnames(data) <- samplenames group1 <- “other” group2 <- “PaC” nTrainingSamples <- 68 nTestSamples <- 35 training <- data[,1:nTrainingSamples ] test <- data[,(nTrainingSamples+1):(nTrainingSamples+nTestSamples)] aprioriBoolean <- is.element(rownames(data) , apri) facTr <- factor(rep( “rest”,ncol(training)),levels=c(group1, group2, “rest”)) subset1Tr <- is.element(groups[1:nTrainingSamples] , group1) subset2Tr <- is.element(groups[1:nTrainingSamples] , group2) facTr[subset1Tr] <- group1 facTr[subset2Tr] <- group2 facTe <- factor(rep( “rest”,ncol(test)),levels=c(group1, group2, “rest”)) subset1Te <- is.element(groups[(nTrainingSamples+1):(nTrainingSamples+nTestSamples)] , strsplit(group1,“,”)[[1]]) subset2Te <- is.element(groups[(nTrainingSamples+1):(nTrainingSamples+nTestSamples)] , strsplit(group2,“,”)[[1]]) facTe[subset1Te] <- group1 facTe[subset2Te] <- group2 svmtrain <- svm(t(training[aprioriBoolean,]) , facTr, kernel=“linear”) pred <- predict(svmtrain, t(test[aprioriBoolean,]) , decision.values = TRUE, probability = T) res <- as.numeric(attributes(pred)$decision.values, probability = T) facnames <- colnames(attributes(pred)$decision.values)[1] ROCdata <- myROC(res,facTe) ROCdata[1] SenSpe <- SensitivitySpecificity(res,facTe) Sensitivity <- as.numeric(SenSpe[,1]) Specificity <- as.numeric(SenSpe[,2]) omSpecificity <- 1-Specificity plot(omSpecificity, Sensitivity, ylab=“Sensitivity”, xlab=“1- Specificity”,type=“1”) mtext(side=1, line = −1.1, paste(“ROC AUC = ”,signif(ROCdata[1], digits=2))) (D) - Final SVM model for PaC vs all (N+ChP+AIP) $call svm.default(x = t(training[aprioriBoolean, ]), y = facTr, kernel = “linear”) $type [1] 0 $kernel [1] 0 $cost [1] 1 $degree [1] 3 $gamma [1] 0.04 $coef0 [1] 0 $nu [1] 0.5 $epsilon [1] 0.1 $sparse [1] FALSE $scaled [1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE $x.scale $x.scale$‘scaled:center’  C1.est..inh. C3..1. C5..1. CD40..2. Eotaxin..3. GM.CSF..1. IgM..B. IL.11..2. IL.12..1. IL.16..1.   24090.45 569451.81 102936.57 22951.29 26674.95 24125.44 20855.98 14129.86 44608.14 20611.42   IL.1a..1. IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3. IL.7..2. Integrin.a.10 MCP.1..3. MCP.3..2. MUC.1..P3.15.   219572.74 19584.88 40985.94 49070.16 24741.71 20879.60 13058.64 11227.79 14915.23 50846.38   Properdin TGF.b1..1. TNF.a..1. TNF.b..1. VEGF..3.   128296.18 22788.14 13682.89 25428.40 41955.64 $x.scale$‘scaled:scale’  C1.est..inh. C3..1. C5..1. CD40..2. Eotaxin..3. GM.CSF..1. IgM..B. IL.11..2. IL.12..1. IL.16..1.   20404.868 122237.943 28461.795 12025.068 13215.275 16954.639 14666.366 10156.372 57988.003 13187.529   IL.1a..1. IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3. IL.7..2. Integrin.a.10 MCP.1..3. MCP.3..2. MUC.1..P3.15.  153112.225 11314.711 76593.575 21019.692 10105.650 19923.025 8856.321 5452.479 6368.842 28650.095  Properdin TGF.b1..1. TNF.a..1. TNF.b..1. VEGF..3.  56720.049 11069.444 8292.061 12180.614 20857.971 $y.scale NULL $nclasses [1] 2 $levels [1] “other” “PaC” “rest” $tot.nSV [1] 27 $nSV [1] 17 10 $labels [1] 1 2 $SV   C1.est..inh. C3..1. C5..1. CD40..2. Eotaxin..3. GM.CSF..1. IgM..B. IL.11..2. IL.12..1. IL.16..1. IL.1a..1. Pa009 −0.75869601 −0.43683015 −0.20743017 −0.005289015 −0.38931564 0.095770897 −0.93670642 −0.05169534 −0.30674506 −0.074417127 0.50792542 Pa038 −0.42410595 −0.73311709 −1.25244853 −0.692015029 −0.05078382 −0.003980786 5.67933331 −0.28192859 −0.28602139 −0.214764177 0.06911333 Pa006 −0.73654816 0.24109661 −0.46040093 −1.012551914 −1.15296122 −0.841258482 −0.72678562 −0.78781462 −0.32368921 −0.848293554 −0.69234178 Pa013 −0.21700833 1.86892400 0.58834639 −0.264721795 −0.68552957 −0.153202159 −0.04927657 0.05757379 −0.08137510 1.221751926 1.08098684 Pa024 1.58648919 1.44742521 0.75444772 −0.284927936 −0.69406449 2.493561060 −0.44527793 −0.39055704 0.29383416 −0.455154737 −0.81120268 Pa056 −0.36431725 −0.46909174 −0.50130198 −0.554747166 −0.35106339 −0.494146089 −0.17013264 −0.60888484 −0.20597864 −0.517666268 1.81952423 Pa125 −0.23467729 0.25849084 0.56420454 0.369299406 0.43426269 0.208651188 1.25308235 −0.20834865 0.08202813 −0.357037439 −0.55800518 Pa001 −0.47076992 0.56381381 0.06633327 0.435287817 0.08932416 −0.222587527 −0.47610845 −0.27857151 −0.35763315 −0.269646986 −0.76364068 Pa010 0.05320001 −0.18419292 −0.59262780 0.130515458 0.29607807 0.356063766 −0.15926715 0.20807177 −0.15588319 0.580881124 −1.35854055 Pa021 0.38072938 −0.51120893 -0.08552594 0.833802541 0.12119111 0.162821416 0.27919151 0.38595265 −0.02303639 0.687902307 −0.40739811 Pa029 −0.39903826 1.23107814 0.03028247 −0.646633317 −0.61805906 −0.141471627 −0.55837741 −0.62561111 −0.08591402 −0.544224442 −0.39352990 Pa048 −0.26957423 0.75584809 −0.08955982 −0.255480195 −0.87836464 −0.745964552 −0.45255194 −0.26172904 0.03931362 −0.449749768 −1.32821555 Pa058 6.10905531 −0.04325893 0.08698841 0.870476656 0.53283461 0.979088486 −0.13691383 −0.05059637 −0.09088942 0.270927629 −0.42939318 Pa092 −0.24149853 −0.22137826 −0.86374589 −0.004986871 −0.21952427 −0.118794005 0.17005557 0.18556909 −0.28585022 0.326046954 −0.87098967 Pa106 −0.23439949 −0.29286642 −0.90705556 1.431821157 0.87865697 1.208446338 −0.20106733 0.67417004 −0.14514808 1.186336278 −0.70633875 Pa128 −0.08417237 0.97358160 0.52585704 −0.062347659 −0.02788034 −0.411255681 0.46178541 −0.17013906 −0.12290537 −0.008370042 −0.89794811 Pa142 −0.50958252 −1.08085583 −1.23272636 −0.299928811 0.33070388 −0.396222532 −0.41999851 −0.39335783 −0.26785141 −0.089558380 −0.27466106 Pa015 −0.71635090 0.14276634 −0.74693179 −0.473760238 −1.26913415 −0.964917983 −0.79188785 −0.58820570 −0.41838792 −0.616645822 0.34550134 Pa025 −0.67904289 1.32071592 −0.29298820 −0.497584785 −0.89584008 −0.363827564 −0.60314141 −0.52579460 −0.42216397 −0.400049252 −0.05516486 Pa027 −0.30026876 0.52136967 −0.53568667 −0.092663002 0.38297396 0.085123350 −0.14805742 −0.08863111 −0.32783954 −0.031648097 −0.41278449 Pa045 2.32770652 1.11229339 2.68850620 −0.053703190 −0.83048219 −0.639849777 −0.14176235 −0.17565594 0.52874104 −0.418407376 0.50156594 Pa047 −0.07301964 −0.60084555−1.39164969 0.347757599 −0.01329602 −0.298201129 0.07255214 0.10285325 −0.08591223 0.019651353 −0.67791456 Pa100 −0.43294548 −0.20545382 −0.28057938 0.237411951 −0.20538311 −0.003113888 0.42568691 −0.47951871 −0.21867907 −0.315650824 −0.68013358 Pa121 −0.05039884 1.52624676 0.54898444 −0.250353390 0.26504091 −0.228812744 −0.10786287 −0.37683608 −0.30183383 −0.169528272 −0.60888710 Pa129 −0.04672091 −1.20455598 −0.72519120 0.144364354 0.39842641 −0.176936684 1.07960964 0.09003457 −0.28256596 0.455159702 −1.07832336 Pa137 −0.81063375 0.39213184 0.40088436 −1.227901001 −1.21269965 −0.956598086 −0.86084278 −0.79973661 7.63985656 −0.907598547 0.72267511 Pa147 −0.13778847 0.76981229 −1.00254399 −0.050061794 0.15942688 −0.300030972 0.29380087 0.02270757 −0.15355286 −0.030274049 0.78409332   IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3. IL.7..2. Integrin.a.10 MCP.1..3. MCP.3..2. MUC.1..P3.15. Properdin TGF.b1..1. Pa009 −0.873764678 −0.411112998 −1.03076857 −0.78714053 −0.5233176752 −1.0569348707 2.27173362 −1.25407017 −1.0252649 0.1360317 −0.2661644 Pa038 −0.206056847 0.009708982 −0.60314751 0.44373161 −0.3919296075 0.8585696202 −0.07305799 −0.03616494 0.8055718 1.6515214 −0.1293500 Pa006 −0.961571105 −0.289056355 −0.34582654 −1.06718063 −0.6638369686 −0.7098872057 −1.23264385 −1.31169599 −0.6500758 −1.3448447 −0.9306547 Pa013 0.234806863 2.175447589 0.01266928 0.54717541 −0.1957777100 −0.4801514210 −0.99759860 −0.91896270 0.6498548 −1.1706810 0.4960250 Pa024 −0.305358326 −0.221631817 0.36267874 −0.18586281 −0.2572215174 −0.0456176001 0.08051256 0.56408243 −0.5609039 −1.1089805 −0.2477406 Pa056 0.217707838 −0.154053300 −0.72632423 −0.17209153 −0.0467640708 −0.5423370601 −0.53963593 −0.61602551 0.9101779 −1.0850042 −0.7841200 Pa125 0.275035954 −0.224926621 −0.11926723 1.08023147 −0.0206201972 −0.0008422566 1.28473810 0.48915923 0.6111125−0.3934791 0.1846749 Pa001 −0.091741993 −0.213105078 −0.47534929 −0.76802149 −0.0327458023 −0.4608177008 −0.98212847 −0.23474026 −0.7966689 −0.2220837 −0.4402696 Pa010 −0.464155570 −0.068647798 0.64754337 −0.22005784 −0.0766204082 0.5695475081 −0.13335331 0.53436185 0.3457089 −0.1103537 −0.2059105 Pa021 0.809674963 0.082111154 0.57592395 0.85625877 0.5975398206 0.3056060393 −0.07617649 1.04456867 2.3882107 0.2397382 0.4214540 Pa029 −0.598511728 −0.226930137 −0.44135325 −0.88287360 −0.4211643576 −0.5463940807 −0.73863658 −0.13480309 −1.0338848 1.3801873 −0.9098994 Pa048 −0.540799012 −0.132230617 −0.48802315 −0.40421411 −0.3511893489 −0.2933086328 −0.69288196 −0.32862131 −0.5117169 −0.5428015 −0.6331645 Pa058 −0.216266096 −0.263744491 −0.02111594 0.02466775 0.7903127709 −0.4266999225 0.04047197 0.59968815 −0.6309139 0.2644843 0.4347455 Pa092 0.188422574 −0.178937815 0.55239494 −0.03520631 0.0359698815 −0.1928812726 −0.60201879 −0.03639941 0.2006243 −0.6324198 −0.2174438 Pa106 0.134678555 −0.023059591 0.13626501 0.32218390 −0.0635055923 −0.0806119478 0.09892621 0.72394826 −0.4466987 0.3305753 0.8539264 Pa128 0.007572306 −0.139367554 0.21470674 −0.12000217 −0.1506709444 0.2875368605 −0.35548071 0.63819849 −0.4067314 1.0337810 0.1537436 Pa142 −0.266469132 −0.281152595 0.82774995 −0.25295292 −0.3165063500 −0.3388735672 1.71680502 0.49242804 −0.2902914 1.5769876 −0.3204796 Pa015 −0.414827646 −0.335150876 −0.59577082 −0.84721879 −0.6165233284 −0.5728379380 −1.06685693 −1.32707320 −0.8710250 −1.3049299 −0.7987132 Pa025 −0.614322842 −0.351470829 −0.93514922 −1.21928380 −0.5784378396 −0.7578714150 −1.07065042 −0.36768309 −1.0996868 −1.1292653 −0.8399010 Pa027 −0.289995023 −0.234718460 −0.34640819 −0.11023567 0.0001702044 0.0004392925 −0.35285696 −0.23601985 0.5504571 −0.8609139 0.5920421 Pa045 −0.071721708 −0.122541540 1.74977696 0.33065121 −0.3344757232 −0.0134402551 0.14558436 −0.40419305 0.5895674 −0.9058004 −0.2084024 Pa047 0.579454777 −0.023082065 1.00137061 −0.02504929 0.3732057511 0.4029526915 0.28311658 0.20414137 −0.3904702 1.3038092 −0.3220880 Pa100 −0.409728805 −0.323942678 0.13563267 −0.32632146 0.2183483192 −0.5858327012 −0.32588781 0.36754365 −0.8761754 1.0424584 −0.3768059 Pa121 −0.177281870 −0.189628137 0.04549011 −0.22387032 0.0960551709 −0.1245495609 −0.01148733 0.08500916 −0.2435519 −0.4560824 −0.2646761 Pa129 0.211284366 −0.255781747 0.20501384 −0.08248423 0.7175913867 0.0496293721 0.39857908 0.87914844 0.9371010 −0.3958375 0.6287985 Pa137 −0.641370977 −0.393683315 −1.25425888 1.23623466 −0.7024639259 −0.6739023588 −0.83664778−0.50259826 −1.1584413 −1.1886815 −1.1372398 Pa147 0.091538371 −0.178739738 0.06208210 0.04644500 −0.0264655443 0.1878050701 0.50826277 −0.60143916 1.5374629 1.3112835 0.0817073   TNF.a..1. TNF.b..1. VEGF..3. Pa009 0.40813630 −0.29072629 0.21376386 Pa038 −0.18826712 0.01047747 −0.29116552 Pa006 −0.66615076 −1.03733849 −0.95344856 Pa013 0.13709356 −0.43789203 −0.39786986 Pa024 0.35985522 −0.69484995 −0.39811383 Pa056 −0.32725609 −0.65537476 −0.81052618 Pa125 0.13099818 0.09666528 0.25745881 Pa001 −0.54730109 −0.26784028 −0.01851188 Pa010 −0.07002966 0.14109466 0.47584857 Pa021 0.35647693 0.84739885 1.41457100 Pa029 −0.70424687 0.01067955 −0.12682955 Pa048 −0.88347271 −0.37892814 0.03951195 Pa058 1.71697123 0.18848291 0.01324853 Pa092 −0.43475610 0.92880031 −0.27996250 Pa106 1.01132754 1.45346413 0.67985051 Pa128 −0.59595956 0.29156722 0.09509325 Pa142 0.12253269 0.41393499 −0.31173738 Pa015 −0.93543929 −0.62606091 −0.82759284 Pa025 −0.16053458 −0.51839963 −0.21614866 Pa027 0.20233072 0.41248908 −0.18909465 Pa045 −0.17202350 −0.50984474 0.77281999 Pa047 −0.29192335 0.22268877 0.33534050 Pa100 0.48915718 −0.30606315 −0.21250898 Pa121 −0.45976438 0.09615171 0.28373940 Pa129 0.29426203 0.62249829 0.08120801 Pa137 −0.91532270 −0.81985007 −1.12040867 Pa147 0.11963137 0.13800477 0.29478773 $index [1] 1 3 15 16 17 21 24 26 28 30 31 32 34 37 38 42 44 47 48 50 53 54 61 64 66 67 68 $rho [1] −0.6668827 $compprob [1] FALSE $probA NULL $probB NULL $sigma NULL $coefs    [,1] [1,] 0.30343126 [2,] 0.09542226 [3,] 1.00000000 [4,] 0.20660644 [5,] 0.05461905 [6,] 0.82341233 [7,] 0.50601183 [8,] 1.00000000 [9,] 0.24168457 [10,] 0.33709008 [11,] 0.15704187 [12,] 0.50405791 [13,] 0.24715788 [14,] 0.85521666 [15,] 0.20152940 [16,] 1.00000000 [17,] 0.50180868 [18,] −1.00000000 [19,] −1.00000000 [20,] −1.00000000 [21,] −0.49831606 [22,] −1.00000000 [23,] −1.00000000 [24,] −1.00000000 [25,] −1.00000000 [26,] −0.10618995 [27,] −0.43058420 $na.action NULL $fitted Pa009 Pa012 Pa038 Pa042 Pa055 Pa063 Pa066 Pa069 Pa081 Pa097 Pa105 Pa108 Pa114 Pa135 Pa006 Pa013 Pa024 Pa033 Pa039 Pa052 Pa056 Pa089 Pa096 Pa125 Pa138 other other other other other other other other other other other other other other other other other other other other other other other other other Pa001 Pa007 Pa010 Pa014 Pa021 Pa029 Pa048 Pa053 Pa058 Pa088 Pa091 Pa092 Pa106 Pa110 Pa119 Pa126 Pa128 Pa133 Pa142 Pa145 Pa005 Pa015 Pa025 Pa026 Pa027 other other other other other other other other other other other other other other other other other other other other PaC other PaC PaC PaC Pa034 Pa040 Pa045 Pa047 Pa070 Pa071 Pa079 Pa080 Pa085 Pa094 Pa100 Pa102 Pa113 Pa121 Pa123 Pa129 Pa137 Pa147  PaC PaC PaC PaC PaC PaC PaC PaC PaC PaC other PaC PaC PaC PaC other PaC PaC Levels: other PaC rest

TABLE VII ROC-AUC values for differentiation between (A) pancreatic cancer, and (B) normal, chronic pancreatitis, and/or acute inflammatory pancreatitis ROC-AUC Biomarker signature 0.71 IL-7 0.69 Integrin α-10 0.76 IL-7 + Integrin α-10 + 1 Table IV B marker 0.79 IL-7 + Integrin α-10 + 2 Table IV B markers 0.80 IL-7 + Integrin α-10 + 3 Table IV B markers 0.79 IL-7 + Integrin α-10 + 4 Table IV B markers 0.81 IL-7 + Integrin α-10 + 5 Table IV B markers 0.81 IL-7 + Integrin α-10 + 6 Table IV B markers 0.80 IL-7 + Integrin α-10 + 7 Table IV B markers 0.84 IL-7 + Integrin α-10 + 8 Table IV B markers 0.79 IL-7 + Integrin α-10 + 9 Table IV B markers 0.80 IL-7 + Integrin α-10 + 10 Table IV B markers 0.79 IL-7 + Integrin α-10 + 11 Table IV B markers 0.76 IL-7 + Integrin α-10 + 12 Table IV B markers The core markers + 8 preferred markers gave the best ROC-AUC value. The signature corresponds to (core marked in red): IL-7 + Integrin α-10 + BTK + C1q + IgM + IL-9 + Procathepsin W + properdin + TM peptide + b-galactosidase However, all marker combinations had substantial predictive power. 

1. A method for determining the presence of pancreatic cancer in an individual comprising or consisting of the steps of: a) providing a sample to be tested from the individual; b) determining a biomarker signature of the test sample by measuring the expression in the test sample of one or more biomarkers selected from the group defined in Table III; wherein the expression in the test sample of one or more biomarkers selected from the group defined in Table III is indicative of the individual having pancreatic cancer; and wherein step (b) comprises or consists of measuring the expression of one or more of the biomarkers listed in Table IV(A) and/or Table IV(B).
 2. The method according to claim 1 further comprising or consisting of the steps of: c) providing a control sample from an individual not afflicted with pancreatic cancer; d) determining a biomarker signature of the control sample by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the presence of pancreatic cancer is identified in the event that the expression in the test sample of the one or more biomarkers measured in step (b) is different from the expression in the control sample of the one or more biomarkers measured in step (d).
 3. The method according to claim 1 or 2 further comprising or consisting of the steps of: e) providing a control sample from an individual afflicted with pancreatic cancer; f) determining a biomarker signature of the control sample by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the presence of pancreatic cancer is identified in the event that the expression in the test sample of the one or more biomarkers measured in step (b) corresponds to the expression in the control sample of the one or more biomarkers measured in step (f).
 4. The method according to claim 1, 2 or 3, wherein step (b) comprises or consists of measuring the expression of one or more of the biomarkers listed in Table IV(A), for example, at least 2 of the biomarkers listed in Table IV(A).
 5. The method according to any one of the preceding claims, wherein step (b) comprises or consists of measuring the expression of interleukin-7 (IL-7) and/or integrin alpha-10, for example, measuring the expression of interleukin-7, measuring the expression of integrin alpha-10, or measuring the expression of interleukin-7 and integrin alpha-10.
 6. The method according to any one of the preceding claims, wherein step (b) comprises or consists of measuring the expression of each the biomarkers listed in Table IV(A).
 7. The method according to any one of the preceding claims, wherein step (b) comprises or consists of measuring the expression of 1 or more of the biomarkers listed in Table IV(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the biomarkers listed in Table IV(B).
 8. The method according to any one of the preceding claims, wherein step (b) comprises or consists of measuring the expression of all of the biomarkers listed in Table IV(B).
 9. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of 1 or more biomarkers from the biomarkers listed in Table IV(C), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 of the biomarkers listed in Table IV(C).
 10. The method according to any one of the preceding claims, wherein step (b) comprises or consists of measuring the expression of all of the biomarkers listed in Table IV(C).
 11. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers defined in Table IV.
 12. The method according to any one of the preceding claims, wherein the method is for differentiating between pancreatic cancer (PaC) and any other disease state or states.
 13. The method according to claim 12, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A).
 14. The method according to claim 12 or 13, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B).
 15. The method according to claim 12, 13 or 14, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in Table V(C).
 16. The method according to any one of claims 12 to 15, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(D), for example at least 2 or 3 of the biomarkers listed in Table V(D).
 17. The method according to any one of claims 12 to 16, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(F), for example at least 2, 3, 4, 5 or 6 of the biomarkers listed in Table V(F).
 18. The method according to any one of claims 12 to 17, wherein step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(C), Table V(D) and/or Table V(F).
 19. The method according to any one of claims 12 to 18, wherein the other disease state or states is chronic pancreatitis (ChP), acute inflammatory pancreatitis (AIP) and/or normal, for example, the other disease state or states may be chronic pancreatitis alone; acute inflammatory pancreatitis alone; chronic pancreatitis and acute inflammatory pancreatitis; chronic pancreatitis and normal; acute inflammatory pancreatitis and normal; or, chronic pancreatitis, acute inflammatory pancreatitis and normal.
 20. The method according to any one of claims 1 to 11, wherein the method is for differentiating between pancreatic cancer and chronic pancreatitis (ChP).
 21. The method according to claim 20, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A).
 22. The method according to claim 20 or 21, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4, or 5 of the biomarkers listed in Table V(C).
 23. The method according to claim 20, 21, or 22, wherein step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A) and/or Table V(C).
 24. The method according to any one of claims 1 to 11, wherein the method is for differentiating between pancreatic cancer and acute inflammatory pancreatitis (AIP).
 25. The method according to claim 24, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A).
 26. The method according to claim 24 or 25, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B).
 27. The method according to claim 24, 25 or 26, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in Table V(C).
 28. The method according to any one of claims 24 to 27, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(E).
 29. The method according to any one of claims 24 to 28, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(F), for example at least 2, 3, 4, 5 or 6 of the biomarkers listed in Table V(F).
 30. The method according to any one of claims 24 to 29, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(H), for example at least 2 or 3 of the biomarkers listed in Table V(H).
 31. The method according to any one of claims 24 to 30, wherein step (b) comprises or consists of or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(C), Table V(E), Table V(F) and/or Table IV(H).
 32. The method according to any one of claims 1 to 11, wherein the method is for differentiating between pancreatic cancer and normal individuals (N).
 33. The method according to claim 32, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A).
 34. The method according to claim 32 or 33, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B).
 35. The method according to claim 32, 33 or 34, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(D), for example at least 2 or 3 of the biomarkers listed in Table V(D).
 36. The method according to any one of claims 32 to 35, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(E).
 37. The method according to any one of claims 32 to 36, wherein step (b) comprises or consists of measuring the expression in the test sample of 1 or more biomarkers from the biomarkers listed in Table V(G), for example at least 2 or 3 of the biomarkers listed in Table V(G).
 38. The method according to any one of claims 32 to 37, wherein step (b) comprises or consists of measuring the expression in the test sample of all of the biomarkers listed in Table V(A), Table V(B), Table V(D), Table V(E) and/or Table IV(G).
 39. The method according to any one of claims 2 to 38, wherein the individual not afflicted with pancreatic cancer is not afflicted with pancreatic cancer (PaC), chronic pancreatitis (ChP) or acute inflammatory pancreatitis (AIP).
 40. The method according to claim 39, wherein the individual not afflicted with pancreatic cancer is not afflicted with any pancreatic disease or condition.
 41. The method according to claim 39 or 40, wherein the individual not afflicted with pancreatic cancer is not afflicted with any disease or condition.
 42. The method according to claim 39, 40 or 41, wherein the individual not afflicted with pancreatic cancer is a healthy individual.
 43. The method according to any one of claims 2 to 38, wherein the individual not afflicted with pancreatic cancer is afflicted with chronic pancreatitis.
 44. The method according to any one of claims 2 to 38, wherein the individual not afflicted with pancreatic cancer is afflicted with acute inflammatory pancreatitis.
 45. The method according to any one of the preceding claims wherein the pancreatic cancer is selected from the group consisting of adenocarcinoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and undifferentiated carcinomas with osteoclast-like giant cells.
 46. The method according to any one of the preceding claims wherein the pancreatic cancer is an adenocarcinoma.
 47. The method according to any one of the preceding claims wherein step (b), (d) and/or step (f) is performed using a first binding agent capable of binding to the one or more biomarkers.
 48. The method according to claim 47 wherein the first binding agent comprises or consists of an antibody or an antigen-binding fragment thereof.
 49. The method according to claim 48 wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.
 50. The method according to claim 48 or 49 wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule.
 51. The method according to any one of claims 48 to 50 wherein the first binding agent is immobilised on a surface.
 52. The method according to any one of claims 1 to 25 wherein the one or more biomarkers in the test sample are labelled with a detectable moiety.
 53. The method according to any one of claims 2 to 25 wherein the one or more biomarkers in the control sample(s) are labelled with a detectable moiety.
 54. The method according to claim 52 or 53 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.
 55. The method according to claim 52 or 53 wherein the detectable moiety is biotin.
 56. The method according to any one of claims 47 to 55 wherein step (b), (d) and/or step (f) is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety.
 57. The method according to any one of claim 56 wherein the second binding agent comprises or consists of an antibody or an antigen-binding fragment thereof.
 58. The method according to claim 57 wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.
 59. The method according to claim 57 or 58 wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule.
 60. The method according to any one of claims 56 to 59 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.
 61. The method according to claim 60 wherein the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g. Alexa647).
 62. The method according to any one of the preceding claims wherein the method comprises or consists of an ELISA (Enzyme Linked Immunosorbent Assay).
 63. The method according to any one of the preceding claims wherein step (b), (d) and/or step (f) is performed using an array.
 64. The method according to claim 63 wherein the array is a bead-based array.
 65. The method according to claim 63 wherein the array is a surface-based array.
 66. The method according to any one of claims 63 to 65 wherein the array is selected from the group consisting of: macroarray; microarray; nanoarray.
 67. The method according to any one of the preceding claims wherein the method comprises: (v) labelling biomarkers present in the sample with biotin; (vi) contacting the biotin-labelled proteins with an array comprising a plurality of scFv immobilised at discrete locations on its surface, the scFv having specificity for one or more of the proteins in Table III; (vii) contacting the immobilised scFv with a streptavidin conjugate comprising a fluorescent dye; and (viii) detecting the presence of the dye at discrete locations on the array surface wherein the expression of the dye on the array surface is indicative of the expression of a biomarker from Table III in the sample.
 68. The method according to any one of claims wherein, step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers.
 69. The method according to claim 68, wherein the nucleic acid molecule is a cDNA molecule or an mRNA molecule.
 70. The method according to claim 68, wherein the nucleic acid molecule is an mRNA molecule.
 71. The method according to claim 68, 69 or 70, wherein measuring the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation.
 72. The method according to any one of claims 68-71, wherein measuring the expression of the one or more biomarker(s) in step (b) is determined using a DNA microarray.
 73. The method according to any one of claims 68 to 72, wherein measuring the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using one or more binding moieties, each individually capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table III.
 74. The method according to claim 73, wherein the one or more binding moieties each comprise or consist of a nucleic acid molecule.
 75. The method according to claim 74 wherein, the one or more binding moieties each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO.
 76. The method according to claim 74 or 75, wherein the one or more binding moieties each comprise or consist of DNA.
 77. The method according to any one of claims 74-76 wherein the one or more binding moieties are 5 to 100 nucleotides in length.
 78. The method according to any one of claims 74-76 wherein the one or more nucleic acid molecules are 15 to 35 nucleotides in length.
 79. The method according to any one of claims 74-78 wherein the binding moiety comprises a detectable moiety.
 80. The method according to claim 79 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety.
 81. The method according to claim 80 wherein the detectable moiety comprises or consists of a radioactive atom.
 82. The method according to claim 81 wherein the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.
 83. The method according to claim 80 wherein the detectable moiety of the binding moiety is a fluorescent moiety.
 84. The method according to any one of the preceding claims wherein, the sample provided in step (b), (d) and/or (f) is selected from the group consisting of unfractionated blood, plasma, serum, tissue fluid, pancreatic tissue, pancreatic juice, bile and urine.
 85. The method according to claim 84, wherein the sample provided in step (b), (d) and/or (f) is selected from the group consisting of unfractionated blood, plasma and serum.
 86. The method according to claim 84 or 85, wherein the sample provided in step (b), (d) and/or (f) is plasma.
 87. An array for determining the presence of pancreatic cancer in an individual comprising one or more binding agent as defined in any one of claims 47 to
 61. 88. An array according to claim 87 wherein the one or more binding agents is capable of binding to all of the proteins defined in Table III.
 89. Use of one or more biomarkers selected from the group defined in Table III as a diagnostic marker for determining the presence of pancreatic cancer in an individual.
 90. The use according to claim 89 wherein all of the proteins defined in Table III are used as a diagnostic marker for determining the presence of pancreatic cancer in an individual.
 91. A kit for determining the presence of pancreatic cancer comprising: C) one or more first binding agent as defined in any one of claims 47 to 55 or an array according to claims 63 to 66 or claim 87 or 88; D) instructions for performing the method as defined in any one of claims 1 to 67 or 68 to
 86. 92. A kit according to claim 31 further comprising a second binding agent as defined in any one of claims 56 to
 61. 93. A method or use for determining the presence of pancreatic cancer in an individual substantially as described herein.
 94. An array or kit for determining the presence of pancreatic cancer in an individual substantially as described herein. 